Antifungal Paints and Coatings

ABSTRACT

Antifungal and antibacterial peptides, polypeptides and proteins as antifungal additives for paint and other coatings are disclosed, along with antifungal compositions, and coated surfaces with antifungal properties. Methods of using the coatings for treating and/or inhibiting growth of mold, mildew and other fungi and bacteria on objects such as building materials that are susceptible to such infestation are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application from prior U.S.patent application Ser. No. 10/884,355 filed Jul. 2, 2004 which claimspriority to U.S. Provisional Application No. 60/485,234 filed Jul. 3,2003.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to antifungal and antibacterialcompositions and methods employing such compositions to deter or preventfungal growth in stored coatings and on susceptible surfaces. Moreparticularly, the present invention relates to such compositionscontaining antifungal and antibacterial peptides, polypeptides orproteins and to methods of making and using such compositions.

2. Description of the Related Art

Fungal growth on indoor and outdoor surfaces is a major environmentalconcern today affecting home, work and recreational environments. Notonly can fungus (e.g., mold, mildew) be unsightly on exposed surfaces,it can destroy wood, fiber and other materials if left untreated,causing severe damage to buildings and other structures and equipment.Over the past few years it has become increasingly apparent thatexposure to certain fungi or their spores can seriously impact thehealth of humans, pets and other animals. Although fungi are certainlynot the only factors that detrimentally affect indoor air quality, inmany instances they have been identified as a primary contributor toindoor air quality problems. In fact, the term “sick building syndrome”was recently coined to describe buildings in which various physical,chemical and biological factors, including growing fungi and/or theirspores, have severely compromised the air quality leading to discomfortor illness of the occupants. Concerns such as allergies, asthma,infections, and the long-term repercussions of mold toxins are just afew of the many real health effects associated with mold contaminationof indoor and outdoor environments.

Fungi (including true fungi, molds and mildews) are eukaryotic organismsthat have cell walls, similar to plants, but do not contain chlorophyll.There are between 100,000-200,000 species of fungi, mold and mildew,depending on which classification methods are used. Of particularconcern are the pathogenic fungi, which can cause significant harm toindividuals who are exposed to them. About 300 species are presentlyknown to be pathogenic for man, but it is thought that there are manyother as yet unrecognized fungal pathogens. The field of medicalmycology has emerged as a result of the growing number of fungal-relatedillnesses and deaths.

Fungi grow as saprophytes, i.e., in a suitable moist environment theyare able to decompose organic matter to obtain the nourishment neededfor growth. Building and decorative materials such as wood, paper-coatedwallboard, wallpaper, fabrics, carpet and leather can provide thenecessary organic matter. Today, an especially problematic fungal genussometimes found in buildings that have excess indoor moisture isStachybotrys. Stachybotrys chartarum, commonly found in nature growingon cellulose-rich plant materials, has often been found in water-damagedbuilding materials, such as ceiling tiles, wallpaper, sheet-rock andcellulose resin wallboard (fiberboard). Depending on the particularconditions of temperature, pH and humidity in which the mold is growing,Stachybotrys may produce mycotoxins, compounds that have toxicproperties.

Other common fungi that can grow in residential and commercial buildingsare Aspergillus species (sp.), Penicillium sp., Fusarium sp., Alternariadianthicola, Aureobasidium pullulans (aka Pullularia pullulans), Phomapigmentivora and Cladosporium sp. The moist indoor environment whichpromotes growth of these fungi can arise from water damage, excessivehumidity, water leaks, condensation, water infiltration, or flooding, insome cases due to defects in building construction, faulty mechanicalsystem design, and/or operational problems. Even modern homes andcommercial buildings are not immune to fungal invasion despite the useof technologically advanced building materials and more energy efficientconstruction and operation than in buildings of the past. Modern homestend to be less well ventilated, and although the use of airconditioning reduces humidity making it harder for mold to grow, today'scentral air conditioning systems can also facilitate the spread of moldspores throughout a home. Increased use of paper products in homes andcommercial buildings today further encourages mold growth. Heavycontamination of indoor or outdoor surfaces by dirt and/or oil can alsoprovide a food source for a fungus. Vulnerable structures and materialsthat are difficult to access for cleaning, or for which cleaning isneglected, are particularly vulnerable to attack by fungi. Fungi arealso known to contaminate stored paints, fuels, and many otherindustrial products.

Fungal colonies typically take on filamentous form, having longfilament-like cells called hyphae. Under the right environmentalconditions, hyphae grow into an intertwining network called themycelium. A mycelium can be visible to the naked eye, appearing asunsightly fuzzy green, bluish-gray or black spots, for example. Whenconditions for growth are less favorable, many varieties of fungi canrespond by forming spores on specialized hyphal cells. Spores are theprimary means for dispersal and survival of fungi, and can remaindormant for months or even years—even withstanding extremely adverseconditions, to germinate and flourish again when environmental variablessuch as light, oxygen levels, temperature, and nutrient availabilityagain become favorable. Thick-walled spores are substantially moreresistant to common disinfective agents than are the thinner-walledvegetative fungal cells. According to the U.S. Environmental ProtectionAgency, there is no practical way to eliminate all mold and mold sporesin the indoor environment.

As mentioned above, paints and paint films or coatings are known to bevulnerable to mold contamination due to the presence of common organiccomponents that act as cellulosic thickeners, surfactants and defoamers,and which can also serve as a source of food for fungus cells. Some ofthese components are casein, acrylic, polyvinyl and other carbonpolymers. For example, latex is a water-dispersed binder comprising acarbon polymer. Inside the paint can, certain fungi (e.g., yeasts) canconvert enough carbon-containing food sources to CO₂ to swell or evenexplode the can. Fungi can also discolor and reduce the viscosity of thepaint, and produce foul odors. Both in-can preservation of paints andprotection of the end use paint films, and the surfaces they cover, frommold, mildew and yeasts is necessary. To combat fungi, a variety ofcoating materials have been formulated which include organic orinorganic chemicals to discourage or prevent the growth of mildew on thepaint film. Ideally, these chemical fungicides or mildewcides slowlyleach out of the paint to the surface, and maintain their inhibitoryproperties for the life of the paint film, causing little or no harm tothe environment. In practice, however, the antifungal properties of mostcoating compositions in use today persist for variable lengths of time,depending on the amount of exposure to the elements, abrasion anderosion.

Most antifungal chemicals are non-specific as to the organism affectedand can be detrimental to the environment, including toxicity to plantand animal life. It is more difficult to identify fungus-specific agentsthan it is to discover bacteria specific-agents because fungal cellsshare many similarities with the cells of higher organisms, whereasbacterial cells are distinctly different. For this reason, fungicidestend to be more toxic to humans and animals than are bactericides. U.S.Pat. No. 5,882,731 (Owens) describes a number of common and proprietarychemical mildewcide-containing products that have been investigated asadditives for water-based latex mixtures. Some known antifungal agentsthat have been used in the coatings industry are: copper (II)8-quinolinolate (CAS No. 10380-28-6); zinc oxide (CAS No. 1314-13-2);zinc-dimethyl dithiocarbamate (CAS No. 137-30-4);2-mercaptobenzothiazole, zinc salt (CAS No. 155-04-4); barium metaborate(CAS No. 13701-59-2); tributyl tin benzoate (CAS No. 4342-36-3); bistributyl tin salicylate (CAS No. 22330-14-9), tributyl tin oxide (CASNo. 56-35-9); parabens: ethyl parahydroxybenzoate (CAS No. 120-47-8),propyl parahydroxybenzoate (CAS No. 94-13-3) methyl parahydroxybenzoate(CAS No. 99-76-3) and butyl parahydroxybenzoate (CAS No. 94-26-8);methylenebis(thiocyanate) (CAS No. 6317-18-6);1,2-benzisothiazoline-3-one (CAS No. 2634-33-5);2-mercaptobenzo-thiazole (CAS No. 149-30-4);5-chloro-2-methyl-3(2H)-isothiazolone (CAS No. 57373-19-0);2-methyl-3(2H)-isothiazolone (CAS No. 57373-20-3); zinc2-pyridinethiol-N-oxide (CAS No. 13463-41-7);tetra-hydro-3,5-di-methyl-2H-1,3,5-thiadiazine-2-thione (CAS No.533-74-4); N-trichloromethyl-thio-4-cyclohexene-1,2-dicarboximide (CASNo. 133-06-2); 2-n-octyl-4-isothiazoline-3-one (CAS No. 26530-20-1);2,4,5,6-tetrachloro-isophthalonitrile (CAS No. 1897-45-6);3-iodo-2-propynyl butylcarbamate (CAS No. 55406-53-6);diiodomethyl-p-tolylsulfone (CAS No. 20018-09-1);N-(trichloromethyl-thio)phthalimide (CAS No. 133-07-3); potassiumN-hydroxy-methyl-N-methyl-dithiocarbamate (CAS No. 51026-28-9); sodium2-pyridinethiol-1-oxide (CAS No. 15922-78-8);2-(thiocyanomethylthio)benzothiazole (CAS No. 21564-17-0);2-4(-thiazolyl) benzimidazole (CAS No. 148-79-8). See V. M. King,“Bactericides, Fungicides, and Algicides,” Ch. 29, pp. 261-267; and D.L. Campbell, “Biological Deterioration of Paint Films,” Ch. 54, pp.654-661; both in PAINT AND COATING TESTING MANUAL, 14^(th) ed. of theGardner-Sward Handbook, J. V. Koleske, Editor (1995), American Societyfor Testing and Materials, Ann Arbor, Mich. Currently, the PesticideAction Network North America (PAN) lists in its Internet chemicaldatabase, www.panna.org, the above-mentioned chemicals plus more than700 additional chemicals designated as pesticides having antifungalproperties in soil and wood.

The mode of action of some of the metal-based antifungal agents isthought to be chelation of metals that are necessary to growth of theorganisms. Some of the nitrogen- and/or sulfur-containing antifungalagents are thought to act by uncoupling oxidative phosphorylation in thefungal cells, or inhibiting oxidation of glucose. The paraben compounds(aka hydroxybenzoate) are thought to affect membrane activity andintegrity.

Due to environmental and safety concerns, there is increasing pressuretoday on the coatings industry to eliminate some of the more effectivebut more toxic chemical preservatives from paints and other coatingcompositions. Yet at the same time, consumers wish to avoid purchasingspoiled or poorly performing products. Thus, there is a great need inthe industry today for safe and effective alternatives to conventionalantifungal agents.

Various naturally occurring biological products that are said to possessantifungal activity are described in the background discussion of U.S.Pat. Nos. 6,020,312; 5,602,097; and 5,885,782 [each incorporated intheir entirety by reference herein]. In many cases, the active componentof those natural antifungal agents has not been identified norcompletely characterized. Since most of the known naturally occurringantifungal agents are poorly characterized at best, the persistence andtoxicity of such compounds in the environment is also unknown.Furthermore, the fact that many of those compounds are produced bymicrobes in the environment suggests that they may have a limitedspectrum of antifungal activity. A drawback of most of the antifungalagents in use today is that they are as toxic to higher organisms asthey are to the target fungi. The more target-specific antifungal agentstend to be very rare and/or costly.

Recently developed methods permit the preparation of synthetic peptidecombinational libraries (“SPCLs”) that are composed of equimolarmixtures of free peptides that can be used with in vitro methods todetermine bioactivity (Furka, A., et al. Int. J. Pept. Protein Res.37:487 (1991), Houghten, R. A., et al. Nature 354:84 (1991), Houghten,R. A., et al. BioTechniques 13:412 (1992). Libraries can consist of D-or L-amino acid stereoisomers or combinations of L- and D- and/ornon-naturally-occurring amino acids. Other methods for synthesizingpeptides of defined sequence are also known. Similarly, large-scalepreparative methods are known. Certain recombinant methods for producingpeptides have also been described, e.g., U.S. Pat. No. 4,935,351. WhileU.S. Pat. Nos. 6,020,312; 5,602,097; and 5,885,782 describe agriculturaluses for certain synthetic antifungal peptides, none of those or anyother peptidic agents have been previously investigated as additives foruse in the paints and coatings industry.

Although significant advancement has been made in identifying variouschemical agents and natural and synthetic peptides or proteins thatdemonstrate antifungal activity for certain uses (e.g., medicaltreatment or agricultural use), there is no indication that any suchbiomolecule could be used successfully in paints or other coatingmaterials for protecting or treating non-living objects. Antifungal orfungus-resistant paints and other coating compositions are needed whichdo not suffer from the same limitations as conventional surface coatingmaterials containing existing fungicides and antifungal agents. Ideally,an antifungal paint will contain fungus-specific fungusdeterring/inhibiting/killing agents that are stable in paints and othercoating mixtures during storage, persist in the resulting coat or filmthat is spread out over a surface in need of protection from fungalinfestation, and which are safer to the environment. Better antifungalmaterials would be especially welcomed by original equipmentmanufacturers (OEMs), and by the architectural, marine and industrialmaintenance industries. In particular, antifungal and antibacterialadditives to paints and coatings that work alone or synergistically withexisting antifungal agents would be desirable.

BRIEF SUMMARY OF THE INVENTION

The compositions and methods of the present invention overcome some ofthe disadvantages of previous antifungal or fungus-resistant paints,coatings and other compositions such as elastomers, textile finishes,adhesives, and sealants. It was not previously known to combine anatural, synthetic or recombinant antifungal peptide, polypeptide orpeptide with a paint or other coating material to provide a coatedsurface with sustainable antifungal activity that protects the recipientsurface from fungal infestation and defacement, and which also providesfungus resistance to the composition itself. It is now disclosed thatantifungal peptidic agents offer a new tool in the arsenal of fungicidaland fungistatic chemicals. Accordingly, new antifungal and antibacterialpaints, coatings, films and other compositions are provided whichcontain one or more bioactive peptides, polypeptides and/or proteins asantifungal and antibacterial peptidic agents. Methods of using theantifungal and antibacterial additives and compositions for treatingexisting fungal or bacterial colonies and/or for deterring or preventingfungal or bacterial infestations and inhibiting cell growth orproliferation on a variety of inanimate objects such as interior andexterior architectural surfaces and building materials are alsoprovided. The compositions and methods disclosed herein avoid many ofthe drawbacks of existing methods and compositions which rely onnon-specific chemical bacteriocides, fungicides, or antifungal agents.By application of a protective coating comprising one or more antifungalor antibacterial protein, polypeptide or peptide, one can prevent ordeter or lessen the infestation and growth of fungus or bacterium. Atthe same time, the associated discoloration, disfiguration and/ordegradation of the supporting substrate or surface can be avoided orreduced. The compositions of the invention are especially useful onsurfaces where conditions are conducive to deposition and development offungus or bacteria, and where control of fungal or bacterial growth ispreferably accomplished with compositions which are not toxic to humans,pets and other animals or harmful to the environment.

In accordance with certain embodiments of the invention, an antifungalcoating composition is provided that is effective for inhibiting thegrowth of Stachybotrys fungi. In some embodiments, an antifungal coatingcomposition is provided that is effective for inhibiting the growth ofone or more of the Aspergillus, Penicillium, Fusarium, Alternaria, andCladosporium genera of fungi. In some embodiments, an antifungal coatingcomposition is effective for inhibiting the growth of Rhizoctonia,Ceratocystis, Pythium, Mycosphaerella, and Candida genera of fungi. Incertain instances, a coating is provided that is either antifungal,antibacterial, or both antifungal and antibacterial.

In certain embodiments of the present invention, compositions areformulated for use as paints and the like, for coating surfaces. Incertain embodiments, such coating includes impregnating porous orsemi-porous materials that are capable of supporting fungal growth.These compositions contain various antifungal peptides or proteins, asdescribed herein, and may also contain chemicals and other substancesthat are conventional and well known in the art. One of the importantbenefits of certain preferred embodiments of the present invention isthat little or no formulation modification, other than the inclusion ofthe presently described antifungal peptide component, is needed toobtain substantial enhancement of fungus resistant, antifungal orfungicidal properties of the coating composition.

A further embodiment comprises using a film or coat comprising thecoating composition, or the composition itself, to protect an object ormaterial selected from the group consisting of wood, paint, adhesive,glue, paper, textile, leather, plastic, cardboard, caulking, frominfestation and growth of a fungus.

A preferred coating material comprises a paint or coating. In someembodiments the paint or coating is applied prophylactically over a“clean” surface that is not contaminated by fungal spores. In otherembodiments the paint or coating is applied to a surface alreadycontaminated by fungal spores or growing fungus.

In certain embodiments of the present invention, a paint or othersurface-coating composition is provided that contains an antifungalprotein, polypeptide or peptide additive that retains its antifungalactivity after being admixed with said paint or other surface-coatingcomposition, and retains antifungal activity after the paint orsurface-coating composition is applied to a surface. Even after drying,the paint or other surface-coating composition renders the coatedsurface antifungal. More specifically, an antifungal paint or othersurface-coating composition comprising antifungal protein, polypeptideor peptide additive is capable of biologically interacting with asusceptible fungus cell or spore in a manner that inhibits or preventsgrowth of the fungus, preferably for extended periods of time. Someadditives of the present invention remained stable in the coating for anextended period of time (e.g., months) at ambient conditions. It iscontemplated that with certain antifungal compositions, especially thosecontaining microencapsulated antifungal peptides, the extended period ofactivity may comprise years. In a preferred embodiment, a polymer-basedcompound that prophalactically and continuously deters fungalinfestation, inhibits or kills fungal cells is provided.

It is contemplated that in certain embodiments the compositions andmethods of the present invention may be used to produce a fungal cellgrowth inhibitory surface, or a fungal cell killing surface, thatremains active for extended periods. Such an antifungal surface may notneed additional treatment with fungicide compositions, clean-uptreatments to effect decontamination and cosmetic painting, therebysimplifying upkeep of the physical condition and appearance of fungusinfestation prone surfaces such as building exteriors. It iscontemplated that in some embodiments the compositions of the presentinvention may be easily applied to susceptible surfaces in advance ofand/or during exposure to a fungus organism.

Isolated naturally occurring proteins, polypeptides and/or peptides areemployed in some embodiments, and in preferred embodiments syntheticproteins, polypeptides and/or peptides are employed. In someembodiments, combinations of natural and/or synthetic proteins,polypeptides and peptides are employed. Still other embodiments employat least one recombinant protein, polypeptide and/or peptide that isproduced using specific expression vectors in a variety of host cells.

Antifungal peptides are chemically defined species that are easilysynthesized and purified. They are not necessarily dependent upon thegenetic stability or growth properties of microorganisms for theirproduction. The methods and compositions of the present invention employan array of different antibiotic compounds which are shown to haveparticular effectiveness in inhibiting the growth of or killing fungalcells. The compositions of the invention are effective in controllingthe growth of fungi, and yet demonstrate a high degree of specificity tothe target fungi, low toxicity and controlled persistence in theenvironment. Using the preferred methods it is possible to produce andidentify desirable antifungal agents for use in paints and coatings in amuch shorter time, and with a considerably higher-probability ofsuccess, than screening natural isolates for antifungal peptides. Sincethe preferred methods of production can control the chemical nature ofthe antifungal agents thus produced, synthesis and purification (ifneeded) of the peptides is much less problematic (e.g., cysteine iseliminated, which amino acid's free sulfhydryl groups can cause unwantedcross linking). Thus, in some embodiments, the paint compositions of thepresent invention comprise peptides of precisely known chemicalstructure and characteristics. The use of D-amino acids increases thestability of certain of these compounds by being insensitive to commonbiological degradation pathways that degrade L-amino acid peptides. Forinstance, L-amino acid peptides may be stabilized by addition of D-aminoacids at one or both of the peptide termini. However, biochemicalpathways are available which will degrade even D-amino acids in thesepeptides so that long-term environmental persistence is not a problem.Of course, where the compositions of the invention act rapidly or neednot otherwise be stabilized, L-amino acids or mixtures of L-and D-aminoacids may be useful. Unlike antifungal agents which only work as one oranother stereoisomer, the compositions of the invention work well aseither one or another stereoisomer or as a mixed stereoisomericcomposition. Research leading to the current invention evaluated SPCLsfor activity against fungal pathogens, including pathogens of plants aswell as those of animals. The library was composed of 52,128,400six-residue peptides, each peptide being composed of D-amino acids andhaving non-acetylated N-termini and amidated C-termini. However, it isnot necessary for a peptide composition of demonstrable antibioticactivity to be completely defined as to each residue. In fact, incertain instances, especially where the peptide compositions of theinvention are being used to treat an array of fungal target organismseach with a different causative agent, mixed peptide compositions willbe preferred. This is also likely to be the case where there is a desireto treat a fungal target with lower concentrations of numerousantifungal additives rather than a higher concentration of a singlechemical composition. In other instances where, for instance, due to theincreased cost of testing or producing a completely defined peptideantibiotic is prohibitive, the mixed peptide compositions of theinvention having one or more variable amino acid residues may bepreferred. In other instances, it may be possible to use peptideantibiotic compositions in coatings that have not been purified, thathave not had their side chains de-blocked, and/or have not been clearedfrom the synthetic resin used to anchor the growing amino acid chains.Thus, antibiotic compositions comprising equimolar mixture of peptidesproduced in a synthetic peptide combinatorial library utilizing themethods of the invention have been derived and shown to have desirableantibiotic activity. In certain embodiments, these relatively variablecompositions are specifically those based upon the sequences of one ormore of the peptides disclosed in any of the U.S. Pat. Nos. 6,020,312;5,602,097; and 5,885,782.

The antibiotic compositions of the invention may also comprise acarrier. In certain instances, the carrier will be one suitable forpermanent surface coating applications. In other instances, the carrierwill be one suitable for use in applying the antibiotic compositions insemi-permanent or temporary coatings. In either instance, the carrierselected should preferably be a carrier whose chemical and/or physicalcharacteristics do not significantly interfere with the antibioticactivity of the peptide composition. It is known, for instance thatcertain microsphere carriers may be effectively utilized withproteinaceous compositions in order to deliver these compositions to asite of preferred activity such as onto a surface. Liposomes may besimilarly utilized to deliver labile antibiotics. Saline solutions,coating-acceptable buffers and solvents and the like may also beutilized as carriers for the peptide compositions of the invention.Those peptides have been demonstrated to inhibit the growth of fungalcells from at least the Fusarium, Rhizoctonia, Ceratocystis, Pythium,Mycosphaerella and Candida species, and are believed to be activeagainst additional genera, including at least some of those that arecapable of infesting building materials and other inanimate objects.

Similarly, processes for inhibiting growth of fungal cells comprisecontacting the fungal cell with a paint or coating compositioncomprising at least one peptide. Using the techniques of the invention,selected pathogenic fungi, some pathogens of plants and others pathogensof animals, have been tested. The processes of the invention have,therefore, been specifically shown to be effective where the fungal cellis a fungal cell selected from the group of fungi consisting of Fusariumand Aspergillus. The processes of the invention are applied to fungalcells of a pathogen of an animal, such as a human. A method forselecting antibiotic compositions is also described. The methodcomprises first creating a synthetic peptide combinatorial library asdescribed herein. Next, as further described in detail herein, a step ofcontacting a battery of fungal cells with aliquots of the syntheticpeptide combinatorial library, each of which aliquots represents anequimolar mixture of peptides in which at least the two C-terminal aminoacid residues are known and which residues are in common for eachpeptide in said mixture is accomplished. After allowing an appropriateperiod for growth, a next step is accomplished in which the growth ofthe battery of fungal cells as compared to untreated control cells ismeasured. Lastly, a determination is made of which of the aliquots mostreduces the growth of fungal cells in a coating overall in the batteryof fungal cells. Of course, the same method may be carried out in whicheach of the aliquots represents an equimolar mixture of peptides inwhich at least three, four, five or more C-terminal amino acid residuesare known (depending upon the overall length of the ultimate peptide inthe SPCL). Typically, such increasingly defined aliquots will besequentially tested in order to select the succeeding best candidatepeptides for testing. Thus, an additional step in the method entailsutilizing the determination of which of the aliquots reduces the growthof fungal cells in a coating overall in said battery of fungal cells toselect which aliquots to next test of a synthetic peptide combinatoriallibrary where at least one additional C-terminal amino acid residue isknown.

A method of treating or preventing growth of a fungus on a susceptiblesurface is also disclosed. These and other objects, features andadvantages of the present invention will be readily apparent to oneskilled in the art from the following detailed description and claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following detailed description, specific examples and claims, whileindicating the preferred embodiments of the invention, are given by wayof illustration only and are considered representative of otherembodiments. Accordingly, it will be readily apparent to one skilled inthe art from this detailed description and the claims which follow thatvarious changes, substitutions and modifications may be made to theinvention disclosed herein without departing from the scope and spiritof the invention.

Paints and other conventional protective or decorative coating materialstypically contain polymeric substances such as casein, acrylic,polyvinyl and carbon polymers (e.g., binders) which can serve asnutrients for fungal cells. As discussed above in the Background of theInvention, not only can these nutrient substances support the growth offungus on paint films or coated surfaces, fungus can also grow insidecans of liquid paints and coating compositions during storage. It was,therefore, an unexpected discovery that certain synthetic peptides, whenadded to a range of conventional paint and coating materials, renderthose compositions resistant to fungal infestation and growth. It wasalso surprising to find that such additives worked alone or inconjunction with existing biocides in a coating.

EXAMPLE 1 Antifungal Peptide Additives for a Coating Composition

A group of preferred antifungal peptides that have either demonstratedactivity as additives for coating mixtures, or that are expected todemonstrate such activity, are disclosed in U.S. Pat. No. 6,020,312(Edwards); U.S. Pat. No. 5,885,782 (Edwards); and U.S. Pat. No.5,602,097 (Edwards), the disclosures of which are hereby incorporated intheir entirety herein by reference. Preferred sequences that will beemployed include one or more of SEQ ID Nos. 1-47, preferably SEQ ID Nos.25-47. These and other peptides with antifungal activity are identifiedusing methods and testing protocols like those described in theabove-referenced patents. Additional peptides that are expected todemonstrate the desired activity in coatings are listed in Table I. Thescreening method generally includes:

-   -   (a) creating a synthetic peptide combinatorial library using        known methods and materials;    -   (b) testing a battery of fungal cells that are known to, or        suspected of, infesting a building material or other object        having a fungus-infestation susceptible surface with aliquots of        the synthetic peptide library, wherein each aliquot comprises an        equimolar mixture of peptides in which at least one of the        C-terminal amino acid residues are known and which residues are        in common for each peptide in the mixture;    -   (c) admixing said aliquots with a coating typically used on such        building material and coating a surface with the admixture;    -   (d) allowing an appropriate period of time for growth of the        fungal cell under suitable culture conditions;    -   (e) comparing the growth of the treated fungal cells with        untreated control cells;    -   (f) identifying which of the aliquots reduced the growth of the        fungal cells; and, optionally, assessing the relative growth        inhibitory activity of each aliquot compared to that of other        aliquots (e.g., comparing IC₅₀ data).

TABLE 1 Seq. Name Source ID Activity Reference Synthetic 1 Fungi U.S.Pat. No. 5,885,782 Synthetic 2 Fungi U.S. Pat. No. 5,885,782 Synthetic 3Fungi U.S. Pat. No. 5,885,782 Synthetic 4 Fungi U.S. Pat. No. 5,885,782Synthetic 5 Fungi U.S. Pat. No. 5,885,782 Synthetic 6 Fungi U.S. Pat.No. 5,885,782 Synthetic 7 Fungi U.S. Pat. No. 5,885,782 Synthetic 8Fungi U.S. Pat. No. 5,885,782 Synthetic 9 Fungi U.S. Pat. No. 5,885,782Synthetic 10 Fungi U.S. Pat. No. 5,885,782 Synthetic 11 Fungi U.S. Pat.No. 5,885,782 Synthetic 12 Fungi U.S. Pat. No. 5,885,782 Synthetic 13Fungi U.S. Pat. No. 5,885,782 Synthetic 14 Fungi U.S. Pat. No. 5,885,782Synthetic 15 Fungi U.S. Pat. No. 5,885,782 Synthetic 16 Fungi U.S. Pat.No. 5,885,782 Synthetic 17 Fungi U.S. Pat. No. 5,885,782 Synthetic 18Fungi U.S. Pat. No. 5,885,782 Synthetic 19 Fungi U.S. Pat. No. 5,885,782Synthetic 20 Fungi U.S. Pat. No. 5,885,782 Synthetic 21 Fungi U.S. Pat.No. 5,885,782 Synthetic 22 Fungi U.S. Pat. No. 5,885,782 Synthetic 23Fungi U.S. Pat. No. 5,885,782 Synthetic 24 Fungi U.S. Pat. No. 5,885,782Synthetic 25 Fungi U.S. Pat. No. 5,885,782 Synthetic 26 Fungi U.S. Pat.No. 5,885,782 Synthetic 27 Fungi U.S. Pat. No. 5,885,782 Synthetic 28Fungi U.S. Pat. No. 5,885,782 Synthetic 29 Fungi U.S. Pat. No. 5,885,782Synthetic 30 Fungi U.S. Pat. No. 5,885,782 Synthetic 31 Fungi U.S. Pat.No. 5,885,782 Synthetic 32 Fungi U.S. Pat. No. 5,885,782 Synthetic 33Fungi U.S. Pat. No. 5,885,782 Synthetic 34 Fungi U.S. Pat. No. 5,885,782Synthetic 35 Fungi U.S. Pat. No. 5,885,782 Synthetic 36 Fungi U.S. Pat.No. 5,885,782 Synthetic 37 Fungi U.S. Pat. No. 5,885,782 Synthetic 38Fungi U.S. Pat. No. 5,885,782 Synthetic 39 Fungi U.S. Pat. No. 5,885,782Synthetic 40 Fungi U.S. Pat. No. 5,885,782 Synthetic 41 Fungi U.S. Pat.No. 5,885,782 Synthetic 42 Fungi U.S. Pat. No. 5,885,782 Synthetic 43Fungi U.S. Pat. No. 5,885,782 Synthetic 44 Fungi U.S. Pat. No. 5,885,782Synthetic 45 Fungi U.S. Pat. No. 5,885,782 Synthetic 46 Fungi U.S. Pat.No. 5,885,782 Synthetic 47 Fungi U.S. Pat. No. 5,885,782 Tachystatin AHorseshoe Crab 48 Gram+ & Gram−, Fujitani (2002) Fungi AndroctoninAndroctonus 49 Gram+ & Gram−, Mandard (1999) Australis Fungi TritrpticinSynthetic 50 Gram+ & Gram−, Schibli (1999) Fungi HNP-3 Defensin Human 51Gram+ & Gram−, Hill (1991) Virus, Fungi Anti-fungal protein Phytolacca52 Fungi Gao (2001) 1 (pafp-s) Americana Magainin 2 Synthetic 53 Gram+ &Gram−, Hara (2001) construct Fungi Indolicidin Bos Taurus 54 Gram+ &Gram−, Rozek (2000) Virus, Fungi Defensin Heliothis 55 Fungi Lamberty(2001) heliomicin virescens Defensin Heliothis 56 Gram+ & Gram−,Lamberty (2001) heliomicin virescens Fungi Sativum defensin 1 Seed ofPea 57 Fungi Almeida (2002) (psd1) Gomesin Synthetic 58 Gram+ & Gram−,Mandard (2002) Fungi, Mammalian cells Lactoferricin B Bovine 59 Gram+ &Gram−, Hwang (1998) Virus, Fungi, Cancer cells PW2 Synthetic 60 FungiTinoco (2002) Hepcidin 20 Human 61 Fungi Hunter (2002) Hepcidin 25 Human62 Fungi Hunter (2002) AC-AMP2 Amaranthus 63 Gram+, Fungi Martins (1996)caudatus NK-Lysin Sus scrofa 64 Gram+ & Gram−, Liepinsh (1997) FungiMagainin 2 African clawed 65 Gram+ & Gram−, Gesell (1997) frog Fungi,cancer cells Melittin B Honey bee 66 Gram+ & Gram−, Eisenberg venomFungi, Mammalian cells Thanatin Podisus 67 Gram+ & Gram−, Mandard (1998)maculiventris Fungi Antimicrobial Common ice 68 Gram+ & Gram−,Michalowski (1998) peptide 1 plant Fungi Melanotropin alpha Bovine 69Gram +, Fungi Cutuli (2000) (Alpha-MSH) CORTICOSTATIN Rabbit 70 Gram+ &Gram−, Selsted (1988) III (MCP-1) Virus, Fungi CORTICOSTATIN Rabbit 71Gram+ & Gram−, Selsted (1988) III (MCP-1) Virus, Fungi Cecropin BChinese oak silk 72 Gram+ & Gram−, Qu (1982) moth Fungi SeminalplasminBovine 73 Gram+ & Gram−, Theil (1983) Fungi, Mammalian cells NP-3Adefensin Rabbit 74 Gram+ & Gram−, Zhu (1992) Virus, Fungi HNP-1 DefensinHuman 75 Gram+ & Gram−, Zhang (1992) Virus, Fungi HNP-2 Defensin Human76 Gram+ & Gram−, Selsted (1989) Virus, Fungi HNP-4 Defensin Human 77Gram+ & Gram−, Wilde (1989) Fungi Histatin 5 Human 78 Gram+ & Gram−, Raj(1998) Fungi Histatin 3 Human 79 Gram+ & Gram−, Oppenheim (1988) FungiHistatin 8 80 Gram+ & Gram−, Yin (2003) Fungi Tracheal Bovine 81 Gram+ &Gram−, Zimmermann (1995) antimicrobial Fungi peptide AMP1 (MJ-AMP1)Garden four- 82 Gram+, Fungi Cammue (1992) o'clock AMP2 (MJ-AMP2) Gardenfour- 83 Gram+, Fungi Cammue (1992) o'clock MBP-1 Maize 84 Gram+ &Gram−, Duvick (1992) Fungi AFP2 Rape 85 Fungi Terras (1993) AFP1 Turnip86 Fungi Terras (1993) AFP2 Turnip 87 Fungi Terras (1993) ADENOREGULINTwo coloured 88 Gram+ & Gram−, Mor (1994) leaf frong Fungi Protegrin 2Pig 89 Gram+ & Gram−, Kokryakov (1993) Virus, Fungi Protegrin 3 Pig 90Gram+ & Gram−, Kokryakov (1993) Virus, Fungi Histatin 1 Crab eating 91Gram+ & Gram−, Xu (1990) macaque Fungi Peptide PGQ African clawed 92Gram+ & Gram−, Moore (1991) frog Fungi Ranalexin Bull frog 93 Gram+ &Gram−, Halverson (2000) Fungi GNCP-2 Guinea pig 94 Gram+ & Gram−,Nagaoka (1991) Virus, Fungi Protegrin 4 Pig 95 Gram+ & Gram−, Zhao(1994) Virus, Fungi Protegrin 5 Pig 96 Gram+ & Gram−, Zhao (1995) Virus,Fungi BMAP-27 Bovine 97 Gram+ & Gram−, Skerlavaj (1996) Fungi BMAP-28Bovine 98 Gram+ & Gram−, Skerlavaj (1996) Fungi Buforin I Asian toad 99Gram+ & Gram−, Park (1996) Fungi Buforin II Asian toad 100 Gram+ &Gram−, Yi (1996) Fungi BMAP-34 Bovine 101 Gram+ & Gram−, Scocchi (1997)Fungi Tricholongin Trichoderma 102 Gram+ & Gram−, Rebuffat (1991)longibrachiatum Fungi Dermaseptin 1 Sauvage's leaf 103 Gram+ & Gram−,Mor (1994) frog Fungi Pseudo-hevein Para rubber tree 104 FungiSoedjanaatmadja (Minor hevin) (1994) Gaegurin-1 Wrinkled frog 105 Gram+& Gram−, Park (1994) Fungi Skin peptide Two-colored leaf 106 Gram+ &Gram−, Mor (1994) tyrosine-tyrosine frog Fungi Penaeidin-1 Penoeidshrimp 107 Gram+ & Gram−, Destoumieux (2000) Fungi Neutrophil Goldenhamster 108 Gram+, Fungi Mak (1996) defensin 1 (HANP- 1) NeutrophilGolden hamster 109 Gram+, Fungi Mak (1996) defensin 3 (HANP- 3) MisgurinOriental 110 Gram+ & Gram−, Park (1997) weatherfish Fungi PN-AMPJapenese morning 111 Gram+, Fungi Koo (1998) glory Histone H2B-1 Rainbowtrout 112 Gram+ & Gram−, Robinette (1998) (HLP-1) Fungi (Fragment)Histone H2b-3 Rainbow trout 113 Fungi Robinette (1998) (HLP-3)(Fragment) Neutrophil Rhesus macaque 114 Gram+ & Gram−, Tang (1999)defensin 2 Fungi (RMAD-2) Termicin Pseudacanthotermes 115 Gram+, FungiLamberty (2001) spiniger Spingerin Pseudacanthotermes 116 Gram+ & Gram−,Lamberty (2001) spiniger Fungi Aurein 1.1 Southern bell 117 Gram+ &Gram−, Rozek (2000) frog Fungi Ponericin G! Ponerine ant 118 Gram+ &Gram−, Orivel (2001) Fungi Brevinin-1BB Rio Grande 119 Gram+ & Gram−,Goraya (2000) leopard frog Fungi Ranalexin-1CB Gree frog 120 Gram+ &Gram−, Halverson (2000) Fungi Ranatuerin-2CA Green frog 121 Gram+ &Gram−, Halverson (2000) Fungi Ranatuerin-2CB Green frog 122 Gram+ &Gram−, Halverson (2000) Fungi Ginkbilobin Ginkgo 123 Gram+ & Gram−, Wang(2000) Virus, Fungi Alpha-basrubrin Malabar spinach 124 Virus, FungiWang (2001) (Fragment) Pseudin 1 Paradoxical frog 125 Gram+ & Gram−,Olson (2001) Fungi Parabutoporin Scorpion 126 Gram+ & Gram−, Moerman(2002) Fungi, Mammalian cells Opistoporin 1 African yellow 127 Gram+ &Gram−, Moerman (2002) leg scorpion Fungi, Mammalian cells Opistoporin 2African yellow 128 Gram+ & Gram−, Moerman (2002) leg scorpion Fungi,Mammalian cells Histone H2A Rainbow trout 129 Gram+, Fungi Fernandes(2002) (fragment) Dolabellanin B2 Sea hare 130 Gram+ & Gram−, Iijima(2002) Fungi Cecropin A Nocutuid moth 131 Gram+ & Gram−, Bulet (2002)Fungi HNP-5 Defensin Human 132 Gram+ & Gram−, Jones (1992) Fungi HNP-6Defensin Human 133 Gram+ & Gram−, Jones (1993) Fungi Holotricin 3Holotrichia 134 Fungi Lee (1995) diomphalia Lingual Bovine 135 Gram+ &Gram−, Schonwetter (1995) antimicrobial Fungi peptide RatNP-3 Rat 136Gram+ & Gram−, Yount (1995) Virus, Fungi GNCP-1 Guinea pig 137 Gram+ &Gram−, Nagaoka (1993) Virus, Fungi Penaeidin-4a Penoeid shrimp 138 Gram+& Gram−, Destoumieux (2000) Fungi Hexapeptide Bovine 139 Gram+ & Gram−,Vogle (2002) Virus, Fungi, Cancer cells P-18 140 Gram+ & Gram−, Lee(2002) Fungi, Cancer cells MUC7 20-Mer Human 141 Gram+ & Gram−, Bobek(2003) Fungi Nigrocin 2 Rana 142 Gram+ & Gram−, Park (2001)nigromaculata Fungi Nigrocin 1 Rana 143 Gram+ & Gram−, Park (2001)nigromaculata Fungi Lactoferrin (Lf) 144 Fungi Ueta (2001) peptide 2Ib-AMP3 Impatiens 145 Gram+, Fungi Ravi (1997) balsamina Ib-AMP4Impatiens 146 Gram+ Fungi Ravi (1997) balsamina Dhvar4 Synthesis 147Gram+ & Gram−, Ruissen (2002) Fungi Dhvar5 Synthesis 148 Gram+ & Gram−,Ruissen (2002) Fungi Synthetic 149 Fungi U.S. App. 10/601,207 Synthetic150 Fungi U.S. App. 10/601,207 Synthetic 151 Fungi U.S. App. 10/601,207Synthetic 152 Fungi U.S. App. 10/601,207 Synthetic 153 Fungi U.S. App.10/601,207 Synthetic 154 Fungi U.S. App. 10/601,207 Synthetic 155 FungiU.S. App. 10/601,207 Synthetic 156 Fungi U.S. App. 10/601,207 Synthetic157 Fungi U.S. App. 10/601,207 Synthetic 158 Fungi U.S. App. 10/601,207Synthetic 159 Fungi U.S. App. 10/601,207 Synthetic 160 Fungi U.S. App.10/601,207 Synthetic 161 Fungi U.S. App. 10/601,207 Synthetic 162 FungiU.S. App. 10/601,207 Synthetic 163 Fungi U.S. App. 10/601,207 Synthetic164 Fungi U.S. App. 10/601,207 Synthetic 165 Fungi U.S. App. 10/601,207Synthetic 166 Fungi U.S. App. 10/601,207 Synthetic 167 Fungi U.S. App.10/601,207 Synthetic 168 Fungi U.S. App. 10/601,207 Synthetic 169 FungiU.S. App. 10/601,207 Synthetic 170 Fungi U.S. App. 10/601,207 Synthetic171 Fungi U.S. App. 10/601,207 Synthetic 172 Fungi U.S. App. 10/601,207Synthetic 173 Fungi U.S. App. 10/601,207 Synthetic 174 Fungi U.S. App.10/601,207 Synthetic 175 Fungi U.S. App. 10/601,207 Synthetic 176 FungiU.S. App. 10/601,207 Synthetic 177 Fungi U.S. App. 10/601,207 Synthetic178 Fungi U.S. App. 10/601,207 Synthetic 179 Fungi U.S. App. 10/601,207Synthetic 180 Fungi U.S. App. 10/601,207 Synthetic 181 Fungi U.S. App.10/601,207 Synthetic 182 Fungi U.S. App. 10/601,207 Synthetic 183 FungiU.S. App. 10/601,207 Synthetic 184 Fungi U.S. App. 10/601,207 Synthetic185 Fungi U.S. App. 10/601,207 Synthetic 186 Fungi U.S. App. 10/601,207Synthetic 187 Fungi U.S. App. 10/601,207 Synthetic 188 Fungi U.S. App.10/601,207 Synthetic 189 Fungi U.S. App. 10/601,207 Synthetic 190 FungiU.S. App. 10/601,207 Synthetic 191 Fungi U.S. App. 10/601,207 Synthetic192 Fungi U.S. App. 10/601,207 Synthetic 193 Fungi U.S. App. 10/601,207Synthetic 194 Fungi U.S. App. 10/601,207 Synthetic 195 Fungi U.S. App.10/601,207 Synthetic 196 Fungi U.S. App. 10/601,207 Synthetic 197 Gram+& Gram−, U.S. App. 10/601,207 Fungi Synthetic 198 Gram+ & Gram−, U.S.App. 10/601,207 Fungi Synthetic 199 Gram+ & Gram−, U.S. App. 10/601,207Fungi

In the above-referenced U.S. Pat. Nos. 6,020,312; 5,885,782; and5,602,097 an iterative process was used to identify active peptidesequences with broad spectrum antifungal activity. A representativemethod employs a hexapeptide library with the first two amino acids ineach peptide chain individually and specifically defined and with thelast four amino acids consisting of equimolar mixtures of 20 aminoacids. Four hundred (400) (20²) different peptide mixtures eachconsisting of 130,321 (19⁴) (cysteine was eliminated) individualhexamers were evaluated. In such a peptide mixture, the finalconcentration for each peptide was 9.38 ng/ml, in a mixture composed of1.5 mg (peptide mix)/ml solution. This mixture profile assumed that anaverage peptide has a molecular weight of 785. This concentration wassufficient to permit testing for antifungal activity. Both D- andL-amino acid containing peptides may be constructed and tested toidentify peptide compositions that can inhibit or kill fungi that cangrow on the surfaces of inanimate objects. Peptide compositionscomprising substantially homogeneous peptide compositions, as well asmixtures of peptides derived from amino acids that are between 3 to 25residues in length (a length readily accomplished using standard peptidesynthesis procedures), especially six residues in length, are disclosedin U.S. Pat. Nos. 6,020,312; 5,885,782; and 5,602,097. A preferredantifungal peptide that inhibits or kills one or more fungus thatinfests and grows on the surfaces of inanimate objects is a hexapeptidehaving the amino acid sequence Phe Arg Leu Lys Phe His (SEQ ID No. 41).

Homogeneous peptide compositions are chiefly composed of a single activepeptide species of a well-defined sequence. Minor amounts (less than 20%by moles) of impurities may coexist with the peptide in thesecompositions so long as they do not interfere with the growth inhibitoryproperties of the active peptide(s). Target fungi include but are notlimited to those fungi that can infest indoor and outdoor structures andbuilding materials causing defacement (e.g., deterioration ordiscoloration), odor, environment hazards, and other undesirableeffects. Alternatively to using one or more isolated antifungal peptidesas the antifungal peptidic agent, the agent may instead be a peptidelibrary aliquot containing a mixture of peptides in which at least two(and preferably three or four) of the N-terminal amino acid residues areknown. If the peptidic agent is a mixture of peptides, at least one willhave antifungal activity. As will be apparent in examples which follow,for ease of production and lower cost, in many instances it will bepreferred to use a peptide library aliquot that contains at least oneantifungal peptide, preferably the hexapeptide of SEQ ID No. 41, but isimpure to the extent that it may also include peptides of unknown exactsequence which may or may not have antifungal activity. In addition, thepeptide or peptide library may be one that has side chains blocked, isattached to the synthetic resin or both blocked and attached.

EXAMPLE 2 Identifying Antifungal Peptides that Inhibit Target Organisms

The testing methods described in U.S. Pat. Nos. 6,020,312; 5,885,782;and 5,602,097 may be employed to screen the peptide library forantifungal activity against a wide variety of fungus genera and species.Preferably the methods are modified to screen against fungal organismsthat are known to, or suspected of, infesting construction materials orother vulnerable materials and surfaces. More preferably, fungal cellsused for screening the peptide library include members of the generaStachybotrys (especially Stachybotrys chartarum), Aspergillus species(sp.), Penicillium sp., Fusarium sp., Alternaria dianthicola,Aureobasidium pullulans (aka Pullularia pullulans), Phoma pigmentivoraand Cladosporium sp. Cell culture conditions may also be modifiedappropriately to provide favorable growth and proliferation conditions,as is within the capability of one of ordinary skill in the art. Theabove-mentioned methods will be used to identify peptides or groups ofpeptides that demonstrate broad-spectrum antifungal activity. Similarmethods will be used to identify particular peptides or groups ofpeptides that target specific fungus genera or species. Alternatively,but less preferred, any other suitable peptide/polypeptide/proteinscreening method could be used instead to identify antifungal peptidecandidates for testing as active antifungal agents in paints and othercoating materials.

It is known that certain of the peptides of particular usefulness in thecoatings of the invention, as disclosed in U.S. Pat. Nos. 6,020,312;5,602,097; and 5,885,782, exhibit variable abilities to inhibit fungalgrowth as adjudged by the minimal inhibitory concentrations (MIC mg/ml)and/or the concentrations necessary to inhibit growth of fifty percentof a population of fungal spores (IC50 mg/ml). MICs may range dependingupon peptide additive and target organism from about 3 to about 300mg/ml, while IC50's may range depending upon peptide additive and targetorganisms from about 2 to about 100 mg/ml. Target organisms susceptibleto these amounts include Fusarium oxysporum, Fusariam Sambucinum,Rhizoctonia Solani, Ceratocystis Fagacearum, Pphiostoma ulmi, Pythiumultimum, Magaporthe Aspergillus nidulans, Aspergillus fumigatus, andAspergillus Parasiticus.

The mode of action of antifungal peptides, polypeptides and proteins, bywhich they exert their inhibitory or fungicidal effects, can be varied.For instance, certain peptides may operate to destabilize fungal cellmembranes, while the modes of action of others could include disruptionsof macromolecular synthesis or metabolism. While the modes of action ofsome known antifungal peptides have been determined (see, e.g., Fiedler,H. P., et al. 1982. Nikomycins: microbial inhibitors of chitin synthase.J. Chem. Technol. Biotechnol. 32:271-280; Isono, K. and S. Suzuki. 1979.The polyoxins: pyrimidine nucleoside peptide antibiotics inhibitingfungal cell wall biosynthesis. Heterocycles 13:333-351), mechanismswhich explain their modes of action and specificity have typically notyet been determined. Initial studies to elucidate antifungal mode ofaction of peptides involves a physical examination of mycelia and cellsto determine if the peptides can perturb membrane functions responsiblefor osmotic balance, as has been observed for other peptides (Zasloff,M. 1987. Proc. Natl. Acad. Sci. USA 84:5449-5453). Disruption ofappressorium formation may also be the mechanism by which some peptidesinhibit fungal growth (see e.g., published U.S. patent application Ser.No. 10/601,207, expressly incorporated herein by reference in itsentirety). For the purposes of preparing and using antifungal peptides,polypeptides and/or proteins as active antifungal agents in paints andother coating compositions, it is not necessary to understand themechanism by which the desired antifungal effect is exerted on funguscells.

EXAMPLE 3 Varying the Amino Acid Sequence of Antifungal Peptides

For the purposes of preparing antifungal paints and other coatingcompositions containing antifungal peptidic agents, it should beappreciated that it is not necessary for the amino acid sequence of apeptide having demonstrable antifungal activity to be completelydefined. In certain situations, especially where an antifungal peptideis being used to target an array of fungal genera or species, mixedpeptide additives may be preferable. This is also likely to be the casewhere there is a desire to treat or prevent infestation by a particularspecies of fungus using lower concentrations of numerous antifungalpeptides rather than a higher concentration of a single peptide. Inother situations where, for instance, due to the increased cost oftesting or producing a completely defined peptide antifungal peptide isprohibitive, the mixed peptide compositions having one or more variableamino acid residues may be preferred. Similarly, it may be preferable toleave synthetic peptides of the invention blocked and/or covalentlyattached to the synthetic resin so long as sufficient antifungalactivity is exhibited in the coating. Thus, antifungal additivecompositions comprising equimolar mixtures of peptides produced in asynthetic peptide combinatorial library utilizing the methods describedherein and/or in U.S. Pat. No. 6,020,312, U.S. Pat. No. 5,885,782, orU.S. Pat. No. 5,602,097 may be employed as antifungal agents in paints,coatings and films.

The antifungal peptide additives for the coatings of the invention maybe constructed using a variety of amino acid precursors. Of course, thepeptides may be homogenous compositions containing only D-, L- or cyclic(non-racemic) amino acids. The chemical structure of such amino acids(which term is used herein to include imino acids), regardless ofstereoisomeric configuration, may be based upon that of the nineteen ortwenty naturally-occurring amino acids: alanine (Ala; A), arginine (Arg;R), asparagine (Asn; N), aspartate (Asp; D), glutamine (Gln; Q),glutamate (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine(Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M),proline (Pro; P), phenylalanine (Phe; F), serine (Ser; S), threonine(Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).Cysteine (Cys; C) is preferably excluded to prevent disulfide linkageproblems in the products. The compositions of the invention may also benon-homogenous, containing for instance both D-, L- and/or cyclic aminoacids. The peptide compositions may also contain amino acids which areother than the naturally-occurring amino acids (e.g., norleucine), asare known to those of skill in the art. The peptides may also beconstructed as retroinversopeptidomimetics of any of the peptides shownto be active in either the D- or L-configurations. It is known, forinstance, that the retroinversopeptidomimetic of SEQ ID No. (41) isinhibitory (albeit less so than either the D- or L-configurations)against certain household fungi such as Fusarium and Aspergillus(Guichard, 1994).

Preferred antifungal paints and coatings will comprise one or more ofthe peptides disclosed in SEQ ID Nos. 1-199, more preferably SEQ ID Nos.1-47. These sequences establish a number of precise chemicalcompositions which have been shown to have antifungal activity against aspectrum of fungi, but which were not previously known to be useful fortreating and/or protecting building materials and other non-livingobjects from infestation by fungi. A highly preferred antifungal peptideis the hexapeptide of SEQ ID No. 41.

In certain instances, the peptides will have completely definedsequences. In other instances, the sequence of the antifungal peptidewill be defined for only certain of the C-terminal amino acid residuesleaving the remaining amino acid residues defined as equimolar ratios.For example, certain of the peptides of SEQ ID Nos. 1-199 have somewhatvariable amino acid compositions. Thus, in each aliquot of the SPCLcontaining a given SEQ ID Nos. having a variable residue, the variableresidues will each be uniformly represented in equimolar amounts by oneof nineteen different naturally-occurring amino acids in one or theother stereoisomeric form. However, the variable residues may be rapidlydefined using the method described in one or more of U.S. Pat. Nos.6,020,312; 5,602,097; and 5,885,782 to identify peptides that possessactivity for controlling fungal growth. In the cited patents it wasdemonstrated that peptides encompassed by the C-terminal sequence“XXXXRF” (SEQ ID No. 1) exhibited antifungal activity for a widespectrum of fungi. For ease of reference, peptides herein are written inthe C-terminal to N-terminal direction to denote the sequence ofsynthesis. However, the conventional N-terminal to C-terminal manner ofreporting amino acid sequences is utilized in the Sequence Listings.This relatively variable composition, therefore, is described as anantifungal peptide even though it is likely that not every peptideencompassed by that general sequence will possess the same or anyantifungal activity.

In the next round of identification of antifungal peptides encompassedby the general sequence “XXXXRF” (SEQ ID No. 1) parent composition ofknown antifungal activity, “XXXLRF” (SEQ ID No. 9) peptides mixtureswere found to exhibit significant antibiotic activity (also disclosed inU.S. Pat. Nos. 6,020,312; 5,602, 097; and 5,885,782). Similarly to theparent composition “XXXXRF” (SEQ ID No. 1), the “XXXLRF” (SEQ ID No. 9)peptides will have a mixed equimolar array of peptides representing thesame nineteen amino acid residues, some of which may have antifungalactivity and some of which may not have such activity. Overall, however,the “XXXLRF” (SEQ ID No. 9) peptide composition is itself an antifungalagent. This process is carried out to the point where completely definedpeptides are produced and tested for their antifungal activity, asdescribed in Example 2 or using any suitable method that would be knownto one of skill in the art. As a result, and as was accomplished for therepresentative peptide “FHLRF” (SEQ ID No. 31), all amino acid residuesin a six residue peptide will be known.

It will be recognized by those of skill in the art that the peptides tobe employed as antifungal agents for paints, coatings and othercompositions, once selected, may be modified to contain functionallyequivalent amino acid substitutions and yet retain the same or similarantifungal characteristics. The importance of the hydropathic index ofamino acids in conferring biological function on a protein has beendiscussed generally by Kyte and Doolittle, J. Mol. Biol., 157:105-132,1982. It is well known that certain amino acids may be substituted forother amino acids having a similar hydropathic index or score and stillretain similar if not identical biological activity. As displayed inTable 2 below, amino acids are assigned a hydropathic index on the basisof their hydrophobicity and charge characteristics. It is believed thatthe relative hydropathic character of the amino acid determines thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with the substrate molecule. Similarly, inpeptides whose secondary structure is not a principal aspect of theinteraction of the peptide, position within the peptide and thecharacteristic of the amino acid residue determine the interactions thepeptide has in a biological system. It is proposed that biologicalfunctional equivalence may typically be maintained where amino acidshaving no more than a +/−1 to 2 difference in the index value, and morepreferably within a +/−1 difference, are exchanged.

TABLE 2 HYDROPATHIC AMINO ACID INDEX Isoleucine 4.5 Valine 4.2 Leucine3.8 Phenylalanine 2.8 Cysteine/Cystine 2.5 Methionine 1.9 Alanine 1.8Glycine −0.4 Threonine −0.7 Tryptophan −0.9 Serine −0.8 Tyrosine −1.3Proline −1.6 Histidine −3.2 Glutamic Acid −3.5 Glutamine −3.5 AsparticAcid −3.5 Asparagine −3.5 Lysine −3.9 Arginine −4.5

Thus, it is expected that isoleucine, for example, which has ahydropathic index of +4.5, can be substituted for valine (+4.2) orleucine (+3.8), and still obtain a protein having similar biologicactivity. Alternatively, at the other end of the scale, lysine (−3.9)can be substituted for arginine (−4.5), and so on. Accordingly, theseamino acid substitutions are generally based on the relative similarityof R-group substituents, for example, in terms of size, electrophiliccharacter, charge, and the like. In general, although these are not theonly such substitutions, the preferred substitutions which take variousof the foregoing characteristics into consideration include thefollowing:

TABLE 3 Originally Screened Residue Exemplary Substitutions alanine gly;ser arginine lys asparagine gln; his aspartate glu cysteine serglutamate asp glutamine asn glycine ala histidine asn; gln isoleucineleu; val leucine ile; val lysine arg; gln; glu methionine met; leu; tyrserine thr threonine ser tryptophan tyr tyrosine trp; phe valine ile;leu

EXAMPLE 4 Stabilized Antifungal Peptide Compositions

A variety of modifications can be made to the peptides as long asantifungal activity is retained. Some modifications may be used toincrease the intrinsic antifungal potency of the peptide. Othermodifications may facilitate handling of the peptide. Peptide functionalgroups that may typically be modified include hydroxyl, amino,guanidinium, carboxyl, amide, phenol, imidazol rings or sulfhydryl.Typical reactions of these groups include but are not limited toacetylation of hydroxyl groups by alkyl halides. Carboxyl groups may beesterified, amidated or reduced to alcohols. Carbodiimides or othercatalysts may be used to catalyze the amidation of carboxyl groups. Theamide groups of asparagine or glutamine may be deamidated under acidicor basic conditions. Acylation, alkylation, arylation or amidationreactions readily occur with amino groups such as the primary aminogroup of the peptide or the amino group of lysine residues. The phenolicgroup of tyrosine can be halogenated or nitrated. Examples wheresolubility of a peptide could be decreased include acylating chargedlysine residues or acetylating the carboxyl groups of aspartic andglutamic acids. Techniques and materials that are suitable for carryingout each of these modifications are well known in the art and have beendescribed in the literature.

Another way in which the antifungal activity of the peptides may bestabilized in paints and other coatings and compositions is by linkingor conjugation to another molecule. Peptides may be conjugated tosoluble or insoluble carrier molecules to modify their solubilityproperties as needed. Examples of soluble carrier molecules includepolymers of polyethyleneglycol and polyvinylpyrrolidone. Alternatively,a peptide may be chemically linked or tethered to an insoluble molecule.Examples of insoluble polymers include sand or other silicates, andpolystyrene, cellulose and polyvinylchloride. Such polymers are oftenemployed in coatings. The molecular size of the conjugated polymerchosen for conjugating with an antifungal peptide is preferably suitedfor carrying out the desired additional function in the coating.Techniques and materials for conjugating peptides to other molecules arewell known in the art and have been described in the literature.

Still another way in which the antifungal activity may be controlled orstabilized is by microencapsulating the peptides to enhance theirstability in liquid coating compositions and in the final paint film orcoat. For example, polyester microspheres may be used to encapsulate andstabilize the peptides in a paint composition during storage, or toprovide for prolonged, gradual release of the peptide after it isdispersed in a paint film covering a surface that is vulnerable toattachment and growth of fungal cells or spores. Any suitablemicroencapsulation technique as would be known to one of ordinary skillin the art may be employed. Such encapsulation may enhance, or confer aparticulate nature to, one or more antifungal peptide. The encapsulatingmembrane may provide protection to the peptide from peptidases,proteases, and other peptide bond or side chain modifying substances, itmay serve to increase the average particle size of the antifungalpeptidic agent to a desired range, and it may allow controlled releaseof the peptide(s) from the encapsulating material, alter surface charge,hydrophobicity, hydrophilicity, solubility and/or dispersability of theparticulate material, or any combination of those functions. Examples ofmicroencapsulation (e.g., microsphere) compositions and techniques aredescribed in Wang, H. T. et al., J. of Controlled Release 17:23-25,1991; and U.S. Pat. Nos. 4,324,683; 4,839,046; 4,988,623; 5,026,650;5,153,131; 6,485,983; 5,627,021 and 6,020,312). Other microencapsulationmethods which may be employed are those described in U.S. Pat. Nos.5,827,531; 6,103,271; and 6,387,399.

EXAMPLE 5 Large-scale Production of Antifungal Peptides

An antifungal peptide sequence identified as described above may begrown in bacterial, insect, or other suitable cells employing techniquesand materials that are well known in the art, except DNA encoding theantifungal peptides described herein will be used instead of a previousDNA sequence. For example, an expression vector will include a DNAsequence encoding SEQ ID No. 1 in the correct orientation and readingframe with respect to the promoter sequence to allow translation of theDNA encoding the SEQ ID No. 1. Examples of the cloning and expression ofan exemplary gene and DNAs are known. Either batch culture productionmethods or continuous fed-batch culture methods may be employed toproduce commercial-scale quantities of antifungal peptides.

EXAMPLE 6 Antifungal Additives Isolated from Microorganisms

Although synthetically obtained antifungal peptidic agents (i. e.,peptides, polypeptides and proteins) that are identified and produced asdescribed above are highly preferred, it is also possible to employsuitable naturally occurring antifungal peptidic agents, and microbesthat produce such agents, as additives in paints and other coatings. Anumber of such naturally occurring peptide additives are listed inTable 1. A drawback to this is the time-consuming process of searchingfor naturally produced antifungal agents with very low-probability ofsuccess. The use of natural antifungal products isolated in commercialquantity from microorganisms is also limited in usefulness due in largepart to purification problems. Large-scale cell culture of theantifungal agent-producing microorganism is required for thepurification of the antifungal product. In many instances, the culturalisolate responsible for the production of the antifungal agent is not anisolate which is easily batch-cultured or it is entirely incapable ofbatch culturing. Furthermore, complicated purification strategies areoften required to purify the active product to a reasonable level ofhomogeneity. A substantial disadvantage to the use of naturally derivedantifungal agents is the potential for co-purification of unwantedmicrobial byproducts, especially byproducts which are undesirably toxic.In many cases, these factors lead to high production costs and makelarge-scale isolation of antifungal products from natural isolatesimpractical. Purifications may be even more difficult where racemizedmixtures are possible where only a single stereoisomer is active, orwhere disulfide linkages are possible between peptide monomers. Evenwhen desirable naturally occurring antifungal proteins or polypeptidesare isolated, for example, and their amino acid sequences at leastpartially identified, synthesis of the native molecule, or portionsthereof, may be problematic due to the need for specific disulfide bondformation, high histidine requirements, and so forth. Nonetheless,natural sources provide additional sequences to be explored as coatingadditives.

EXAMPLE 7 Coating Formulations Containing Antifungal Peptide Additives

One or more of the antifungal peptides or peptide compositions, preparedas described in any of the foregoing examples, is mixed with a basepaint or other coating, which may be any suitable commercially availableproduct, a wide variety of which are well known in the art. Preferablythe base composition is free of chemicals and other additives that aretoxic to humans or animals, and/or that fail to comply with applicableenvironmental safety rules or guidelines. In some instances, it may bepreferred to custom blend a paint or coating mixture using anycombination of various naturally-occurring and synthetic components andadditives that are known in the art and are also described in U.S.patent application Ser. No. 10/655,345 filed Sep. 4, 2003 or U.S. patentapplication Ser. No. 10/792,516 filed on Mar. 3, 2004, which are herebyexpressly incorporated herein by reference in their entirety.

Coating components generally include a binder, a liquid component, acolorizing agent, one or more additive, or a combination of any ofthose. A coating typically comprises a material often referred to as a“binder,” which is the primary material in a coating capable ofproducing a film. In most embodiments, a coating will comprise a liquidcomponent (e.g., a solvent, a diluent, a thinner), which often confersand/or alters the coating's rheological properties (e.g., viscosity) toease the application of the coating to a surface. Usually a coating(e.g., a paint) will comprise a colorizing agent (e.g., a pigment),which usually functions to alter an optical property of a coating and/orfilm. A coating will often comprise an additive, which is a compositionincorporated into a coating to (a) reduce and/or prevent the developmentof a physical, chemical, and/or aesthetic defect in the coating and/orfilm; (b) confer some additional desired property to a coating and/orfilm; or (c) a combination thereof. Examples of an additive include anaccelerator, an adhesion promoter, an antifloating agent, anantiflooding agent, an antifoaming agent, an antioxidant, anantiskinning agent, a buffer, a catalyst, a coalescing agent, acorrosion inhibitor, a defoamer, a dehydrator, a dispersant, a drier, anelectrical additive, an emulsifier, a film-formation promoter, a fireretardant, a flow control agent, a gloss aid, a leveling agent, a lightstabilizer, a marproofing agent, a matting agent, a neutralizing agent,a preservative, a rheology modifier, a slip agent, a viscosity controlagent, a wetting agent, or a combination thereof. The content for anindividual coating additive in a coating generally is 0.0001% to 20.0%,including all intermediate ranges and combinations thereof. However, inmany instances it is preferred if the concentration of a single additivein a coating comprises between 0.0001% and 10.0%, including allintermediate ranges and combinations thereof.

Some of the usual types of components of paints and coatings aresummarized as follows:

Binders: oil-based (e.g., oils, alkyd resins, oleoresinous binders, andfatty acid epoxy esters; polyester resins; modified cellulose;polyamide; amidoamine; amino resins; urethanes; phenolic resins; epoxyresins; polyhydroxyether; acrylic resins; polyvinyl binders; rubberresins; bituminous; polysulfide and silicone.

Liquid Components: solvents; thinners; diluents; plasticizers; and water(e.g., hydrocarbons; oxygenated solvents; chlorinated hydrocarbons,nitrated hydrocarbons, other organic liquids)

Colorants: pigments and dyes.

Additives: preservatives (e.g.,biocides/bactericides/fungicides/algaecides); wetting agents; buffers(e.g., ammonium bicarbonate, both monobasic and dibasicphosphatebuffers, Trizma base and zwitterionic buffers); rheology modifiers;defoamers; catalysts (e.g., driers, acids, bases, urethane catalysts);antiskinning agents; light stabilizers; corrosion inhibitors;dehydrators; electrical additives; and anti-insect additives.

Preservatives serve to reduce or prevent the deterioration of a coatingand/or film by a microorganism, by acting as a biocide, which kills anorganism, a biostatic, which reduces or prevents the growth of anorganism, or a combination of effects. Examples of a biocide include,for example, a bactericide, a fungicide, an algaecide, or a combinationthereof.

A preferred paint or coating composition contains, as a preservative, anantifungal peptidic agent (i.e., one or more peptides, polypeptides orproteins), according to any of Examples 1-6. An antifungal peptidicagent may be used as a partial or complete substitute (“replacement”)for another fungicide and/or fungistatic that is typically used in afungus prone composition. It is contemplated that 0.0001% to 100%,including all intermediate ranges and combinations thereof, of aconventional antifungal component in a coating formulation may besubstituted by an antifungal peptidic agent. In some formulations, theconcentration of antifungal peptidic agent may exceed 100%, by weight orvolume, of the non-peptidic antifungal component (fungicide orfungistat) that is being replaced. A conventional non-peptidicantifungal component may be replaced with an antifungal peptidic agentequivalent to 0.001% to 500% (by weight, or by volume), including allintermediate ranges and combinations thereof, of the substitutedantifungal component. For example, to produce a coating with similarfungal resistance properties as a non-substituted formulation, it mayrequire that 20% (e.g., 0.2 kg) of a chemical fungicide may be replacedby 10% (e.g., 0.1 kg) of an antifungal peptidic agent. In anotherexemplary formulation, to produce a coating with similar fungalresistance as a non-substituted formulation, it may require replacing70% of a chemical fungicide (e.g., 0.7 kg) with the equivalent of 127%(e.g., 1.27 kg) of antifungal peptidic agent. The various assaysdescribed herein, or as would be known to one of ordinary skill in theart in light of the present disclosure, may be used to determine thefungal resistance properties of a composition (e.g., a coating, a film)produced by direct addition of an antifungal peptidic agent and/orsubstitution of some or all of a non-peptidic or chemical antifungalcomponent by an antifungal peptidic agent. Such additives may bedirectly admixed with the coating, applied as a primer coating, appliedas an overcoat, or any combination of these application techniques.

A preservative may comprise an in-can preservative, an in-filmpreservative, or a combination thereof. An in-can preservative is acomposition that reduces or prevents the growth of a microorganism priorto film formation. Addition of an in-can preservative during awater-borne coating production typically occurs with the introduction ofwater to a coating composition. Typically, an in-can preservative isadded to a coating composition for function during coating preparation,storage, or a combination thereof. An in-film preservative is acomposition that reduces or prevents the growth of a microorganism afterfilm formation. Oftentimes an in-film preservative is the same chemicalas an in-can preservative, but added to a coating composition at ahigher (e.g., two-fold) concentration for continuing activity after filmformation.

Examples of preservatives that have been used in coatings include ametal compound (e.g., an organo-metal compound) biocide, an organicbiocide, or a combination thereof. Examples of a metal compound biocideinclude barium metaborate (CAS No. 13701-59-2), which is a fungicide andbactericide; copper (II) 8-quinolinolate (CAS No. 10380-28-6), which isa fungicide; phenylmercuric acetate (CAS No. 62-38-4), tributyltin oxide(CAS No. 56-35-9), which is less preferred for use against Gram-negativebacteria; tributyltin benzoate (CAS No. 4342-36-3), which is a fungicideand bactericide; tributyltin salicylate (CAS No. 4342-30-7), which is afungicide; zinc 2-pyridinethiol-N-oxide (CAS No. 13463-41-7), which is afungicide; zinc oxide (CAS No. 1314-13-2), which is afungistatic/fungicide and algaecide; a combination ofzinc-dimethyldithiocarbamate (CAS No. 137-30-4) and zinc2-mercaptobenzothiazole (CAS No. 155-04-4), which acts as a fungicide;zinc 2-pyridinethiol-N-oxide (CAS No. 13463-41-7), which is a fungicide;a metal soap; or a combination thereof. Examples of metals comprised ina metal soap biocide include copper, mercury, tin, zinc, or acombination thereof. Examples of an organic acid comprised in a metalsoap biocide include a butyl oxide, a laurate, a naphthenate, anoctoate, a phenyl acetate, a phenyl oleate, or a combination thereof. Itis anticipated that the peptide additives of the present invention willwork in combination with or synergistically with such preservatives.

An example of an organic biocide that acts as an algaecide includes2-methylthio-4-tert-butylamino-6-cyclopropylamino-s-triazine (CAS No.28159-98-0). Examples of an organic biocide that acts as a bactericideinclude a combination of 4,4-dimethyl-oxazolidine (CAS No. 51200-87-4)and 3,4,4-trimethyloxazolidine (CAS No. 75673-43-7);5-hydroxy-methyl-1-aza-3,7-dioxabicylco (3.3.0.) octane (CAS No.59720-42-2); 2(hydroxymethyl)-aminoethanol (CAS No. 34375-28-5);2-(hydroxymethyl)-amino-2-methyl-1-propanol (CAS No. 52299-20-4);hexahydro-1,3,5-triethyl-s-triazine (CAS No. 108-74-7);1-(3-chloroallyl)-3,5,7-triaza-1-azonia-adamantane chloride (CAS No.51229-78-8); 1-methyl-3,5,7-triaza-1-azonia-adamantane chloride (CAS No.76902-90-4); p-chloro-m-cresol (CAS No. 59-50-7); an alkylaminehydrochloride; 6-acetoxy-2,4-dimethyl-1,3-dioxane (CAS No. 828-00-2);5-chloro-2-methyl-4-isothiazolin-3-one (CAS No. 26172-55-4);2-methyl-4-isothiazolin-3-one (CAS No. 2682-20-4);1,3-bis(hydroxymethyl)-5,5-dimethylhydantoin (CAS No. 6440-58-0);hydroxymethyl-5,5-dimethylhydantoin (CAS No. 27636-82-4); or acombination thereof. Examples of an organic biocide that acts as afungicide include a parabens; 2-(4-thiazolyl)benzimidazole (CAS No.148-79-8); N-trichloromethyl-thio-4-cyclohexene-1,2-dicarboximide (CASNo. 133-06-2); 2-n-octyl-4-isothiazoline-3-one (CAS No. 26530-20-1);2,4,5,6-tetrachloro-isophthalonitrile (CAS No. 1897-45-6);3-iodo-2-propynyl butyl carbamate (Cas No. 55406-53-6);N-(trichloromethyl-thio)phthalimide (Cas No. 133-07-3);tetrachloroisophthalonitrile (Cas No. 1897-45-6); potassiumN-hydroxy-methyl-N-methyl-dithiocarbamate (Cas No. 51026-28-9); sodium2-pyridinethiol-1-oxide (Cas No. 15922-78-8); or a combination thereof.Examples of a parbens include butyl parahydroxybenzoate (Cas No.94-26-8); ethyl parahydroxybenzoate (Cas No. 120-47-8); methylparahydroxybenzoate (Cas No. 99-76-3); propyl parahydroxybenzoate (CasNo. 94-13-3); or a combination thereof. Examples of an organic biocidethat acts as an bactericide and fungicide include2-mercaptobenzo-thiazole (Cas No. 149-30-4); a combination of5-chloro-2-methyl-3(2H)-isothiazoline (Cas No. 26172-55-4) and2-methyl-3(2H)-isothiazolone (Cas No. 2682-20-4); a combination of4-(2-nitrobutyl)-morpholine (Cas No. 2224-44-4) and4,4′-(2-ethylnitrotrimethylene dimorpholine (Cas No. 1854-23-5);tetra-hydro-3,5-di-methyl-2H-1,3,5-thiadiazine-2-thione (Cas No.533-74-4); potassium dimethyldithiocarbamate (Cas No. 128-03-0); or acombination thereof. An example of an organic biocide that acts as analgaecide and fungicide includes diiodomethyl-p-tolysulfone (Cas No.20018-09-1). Examples of an organic biocide that acts as an algaecide,bactericide and fungicide include glutaraldehyde (CAS No. 111-30-8);methylenebis(thiocyanate) (Cas No. 6317-18-6);1,2-dibromo-2,4-dicyanobutane (CAS No. 35691-65-7);1,2-benzisothiazoline-3-one (CAS No. 2634-33-5);2-(thiocyanomethyl-thio)benzothiazole (CAS No. 21564-17-0); or acombination thereof. An example of an organic biocide that acts as analgaecide, bactericide, fungicide and molluskicide includes2-(thiocyanomethyl-thio)benzothiozole (CAS No. 21564-17-0) and methylenebis(thiocyanate) (CAS No. 6317-18-6).

In certain situations of use, an applicable environmental law orregulation may encourage the selection of an organic biocide such as abenzisothiazolinone derivative. An example of a benzisothiazolinonederivative is Busan™ 1264 (Buckman Laboratories, Inc.), Proxel™ GXL(Avecia Inc.), or Preventol® VP OC 3068 (Bayer Corporation), whichcomprises 1,2-benzisothiazolinone (CAS No. 2634-33-5). In the case ofBusan™ 1264, the primary use is a bactericide and/or fungicide at 0.03%to 0.5% in a water-borne coating.

Often, a preservative is a proprietary commercial formulation and/or acompound sold under a tradename. Examples include organic biocides underthe tradename Nuosept® (International Specialty Products), which aretypically used in a water-borne coating. Specific examples of a Nuosept®biocide includes Nuosept® 95, which comprises a mixture of bicyclicoxazolidines, and is typically added to 0.2% to 0.3% concentration to acoating composition; Nuosept® 145, which comprises an amine reactionproduct, and is typically added to 0.2% to 0.3% concentration to acoating composition; Nuosept® 166, which comprises4,4-dimethyloxazolidine (CAS No. 51200-87-4), and is typically added to0.2% to 0.3% concentration to a basic pH water-borne coatingcomposition; or a combination thereof. A further example is Nuocide®(International Specialty Products) biocides, which are typically usedfungicides and/or algaecides. Examples of a Nuocide® biocide is Nuocide®960, which comprises 96% tetrachlorisophthalonitrile (CAS No.1897-45-6), and is typically used at 0.5% to 1.2% in a water-borne orsolvent-borne coating as a fungicide; Nuocide® 2010, which compriseschlorothalonil (CAS No. 1897-45-6) and IPBC (CAS No. 55406-53-6) at 30%,and is typically used at 0.5% to 2.5% in a coating as a fungicide andalgaecide; Nuocid® 1051 and Nuocide® 1071, each which comprises 96%N-cyclopropyl-N-(1-dimethylethyl)-6-(methylthio)-1,3,5-triazine-2,4-diamine(CAS No. 28159-98-0), and is typically used as an algaecide inantifouling coatings at 1.0% to 6.0% or water-based coatings at 0.05% to0.2%, respectively; and Nuocide® 2002, which comprises chlorothalonil(CAS No. 1897-45-6) and a triazine compound at 30%, and is typicallyused at 0.5% to 2.5% in a coating and/or a film as a fungicide andalgaecide.

An additional example of a tradename biocide for coatings includesVancide® (R. T. Vanderbilt Company, Inc.). Examples of a Vancide®biocide include Vancide® TH, which compriseshexahydro-1,3,5-triethyl-s-triazine (CAS No. 108-74-7), and is generallyused in a water-borne coating; Vancide® 89, which comprisesN-trichloromethylthio-4-cyclohexene-1,2-dicarboximide (CAS No. 133-06-2)and related compounds such as captan (CAS No. 133-06-2), and is used asa fungicide in a coating composition; or a combination thereof. Abactericide and/or fungicide for coatings, particularly a water-bornecoating, is a Dowicil™ (Dow Chemical Company). Examples of a Dowicil™biocide include Dowicil™ QK-20, which comprises2,2-dibromo-3-nitrilopropionamide (CAS No. 10222-01-2), and is used as abactericide at 100 ppm to 2000 ppm in a coating; Dowicil™ 75, whichcomprises 1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride(CAS No. 51229-78-8), and is used as a bactericide at 500 ppm to 1500ppm in a coating; Dowicil™ 96, which comprises 7-ethylbicyclooxazolidine (CAS No. 7747-35-5), and is used as a bactericide at1000 ppm to 2500 ppm in a coating; Bioban™ CS-1135, which comprises4,4-dimethyloxazolidine (CAS No. 51200-87-4), and is used as abactericide at 100 ppm to 500 ppm in a coating; or a combinationthereof. An additional example of a tradename biocide for coatingsincludes Kathon® (Rohm and Haas Company). An example of a Kathon®biocide includes Kathon® LX, which typically comprises5-chloro-2-methyl-4-isothiazolin-3-one (CAS no 26172-55-4) and2-methyl-4-isothiazolin-3-one (CAS no 2682-20-4) at 1.5%, and is addedfrom 0.05% to 0.15% in a coating. Examples of tradename fungicides andalgaecides include those described for Fungitrol® (InternationalSpecialty Products), which are often formulated for solvent-borne andwater-borne coatings, and in-can and film preservation. An example isFungitrol® 158, which comprises 15% tributyltin benzoate (CAS No.4342-36-3) (15%) and 21.2% alkylamine hydrochlorides, and is typicallyused at 0.35% to 0.75% in a water-borne coating for in-can and filmpreservation. An additional example is Fungitrol® 11, which comprisesN-(trichloromethylthio)phthalimide (CAS No. 133-07-3), and is typicallyused at 0.5% to 1.0% as a fungicide for solvent-borne coating. A furtherexample is Fungitrol® 400, which comprises 98% 3-iodo-2-propynl N-butylcarbamate (“IPBC”) (Cas No. 55406-53-6), and is typically used at 0.15%to 0.45% as a fungicide for a water-borne or a solvent-borne coating.See Table 4.

TABLE 4 Company Product Roster Arch Chemicals, Inc. Zinc Ornadine (ZincPyrithione/fungicide/algaecide) 800.344.9168/Fax: 203.271.4060 SodiumOmadine (Sodium Pyrithione/fungicide- E-mail: sales@archbiocides.comalgaecide) www.archbiocides.com Copper Omadine (CopperPyrithione/algaecide) Triadine 174 (Triazine/bactericide) Omacide IPBC(Iodopropynyl-butyl carbomate/fungicide) Other: antifouling agentsAvecia Protection & Hygiene Proxel GXL (BIT) Wilmington, DE Proxel BDZO(BIT) 800.523.7391/Fax: 302.477.8120 Proxel BZ (BIT/ZPT) E-mail:biocides@avecia.com Proxel TN (BIT/Triazine) www.avecia.com/biocidesProxel XL2 (BIT) Densil C404 (Chlorthalonil) Densil P (Densil P) DensilDN (BUBIT) Vantocil IB (PHMB) BASF Corp. Myacide AS Technical (Bronopol,solid) Mount Olive, NJ Myacide AS 2, 30 and 15 (Bronopol, solutions)973.426.4358/Fax: 973.426.3863 Myacide GDA Technical (50%Glutaraldehyde) www.biocides.basf-corp.com Myacide GA 42, 26 & 15(Glutaraldehyde, solut.) Protectol PE (Phenoxyethanol, liquid) DaometTechnical (Dazoment, solid) Myacide HT Technical (Triazine, liquid)Buckman Laboratories, Inc. Busan (wood preservative/pkg. pres.) Memphis,TN Butrol (corrosion inhibitor/rust inhibitor) E-mail:knetix@buckman.com Busan (bactericide; mold inhibitor and biocide)Cognis Corp. Nopcocide N400 (Cholorthalonil-40% solution) Ambler, PANopcocide N-98 (Chlorothalonil-100%) 800.445.2207/Fax: 215.628.1111Nopcocide P-20 (IPBC-20% solution) E-mail: Nopcocide P-40 (IPBC-40%solution) shruti.singhal@cognis.com Nopcocide P-100 (IPBC-100% active)www.cognis.com International Specialty Products Fungitrol (fungicides)aka ISP Biotrend (biocides) Wayne, NJ Nuocide (fungicides/algaecides)800.622.4423/Fax: 973.628.4001 Nuosept (antimicrobial agents) E-mail:info@ispcorp.com www.ispcorp.com Rohm and Haas Company Katon LX 1.5%(preservative) Philadelphia, PA Rocima 550 (preservative)www.rohmhaas.com Rocima 607 (preservative) Rozone 2000 (dry filmfungicide) Skane M-8 (dry film fungicide) Troy Corp. Polyphase 678Florham Park, NJ Polyphase 663 Fax: 973.443.0843 Polyphase CST E-mail:marketing@troycorp.com Polyphase 641 www.troycorp.com Troysan 680 MergalK10N

As would be known to one of ordinary skill in the art, determination ofwhether damage to a coating and/or film is due to microorganisms (e.g.,film algal defacement, film fungal defacement), as well as the efficacyof addition of a preservative to a coating and/or film composition inreducing microbial damage to a coating and/or film, may be empiricallydetermined by techniques such as those that are described in “ASTM Bookof Standards, Volume 06.01, Paint—Tests for Chemical, Physical, andOptical Properties; Appearance,” D3274-95, D4610-98, D2574-00, D3273-00,D3456-86, D5589-97, and D5590-00, 2002; and in “Paint and CoatingTesting Manual, Fourteenth Edition of the Gardner-Sward Handbook,”(Koleske, J. V. Ed.), pp. 654-661, 1995. Examples of microorganismstypically selected in such procedures as positive controls of a coatingand/or film damaging microorganism include, for example, Aspergillusoryzae (ATCC 10196), Aspergillus flavus (ATCC 9643), Aspergillus niger(ATCC 9642), Pseudomonas aeruginosa (ATCC 10145), Aureobasidiumpullulans (ATCC 9348), Penicillium citrinum (ATCC 9849), Penicilliumfuniculosum (ATCC 9644), or a combination thereof.

In general, a preservative, and use of a preservative in a coating, isknown to those of skill in the art, and all such materials andtechniques for using a preservative in a coating may be applied in thepractice of the present invention (see, for example, Flick, E. W.“Handbook of Paint Raw Materials, Second Edition,” 263-285 and 879-998,1989; in “Paint and Coating Testing Manual, Fourteenth Edition of theGardner-Sward Handbook,” (Koleske, J. V. Ed.), pp 261-267 and 654-661,1995; in “Paint and Surface Coatings, Theory and Practice, SecondEdition,” (Lambourne, R. and Strivens, T. A., Eds.), pp. 193-194,371-382 and 543-547, 1999; Wicks, Jr., Z. W., Jones, F. N., Pappas, S.P. “Organic Coatings, Science and Technology, Volume 1: Film Formation,Components, and Appearance,” pp. 318-320, 1992; Wicks, Jr., Z. W.,Jones, F. N., Pappas, S. P. “Organic Coatings, Science and Technology,Volume 2: Applications, Properties and Performance,” pp. 145, 309,319-323 and 340-341, 1992; in “Paints, Coatings and Solvents, Second,Completely Revised Edition,” (Stoye, D. and Freitag, W., Eds.) pp 6, 127and 165, 1998; and in “Handbook of Coatings Additives”, pp. 177-224,1987).

It is contemplated that any previously described formulation of afungal-prone composition may be modified to incorporate an antifungalpeptidic agent. Examples of described coating compositions include over200 industrial water-borne coating formulations (e.g., air dry coatings,air dry or force air dry coatings, anti-skid of non-slip coatings, bakedry coatings, clear coatings, coil coatings, concrete coatings, dippingenamels, lacquers, primers, protective coatings, spray enamels, trafficand airfield coatings) described in “Industrial water-based paintformulations,” 1988, over 550 architectural water-borne coatingformulations (e.g., exterior paints, exterior enamels, exteriorcoatings, interior paints, interior enamels, interior coatings,exterior/interior paints, exterior/interior enamels, exterior/interiorprimers, exterior/interior stains), described in “Water-based tradepaint formulations,” 1988, the over 400 solvent borne coatingformulations (e.g., exterior paints, exterior enamels, exteriorcoatings, exterior sealers, exterior fillers, exterior primers, interiorpaints, interior enamels, interior coatings, interior primers,exterior/interior paints, exterior/interior enamels, exterior/interiorcoatings, exterior/interior varnishes) described in “Solvent-based paintformulations,” 1977; and the over 1500 prepaint specialties and/orsurface tolerant coatings (e.g., fillers, sealers, rust preventives,galvanizers, caulks, grouts, glazes, phosphatizers, corrosioninhibitors, neutralizers, graffiti removers, floor surfacers) describedin Prepaint Specialties and Surface Tolerant Coatings, by Ernest W.Flick, Noyes Publications, 1991.

An exemplary exterior gloss alkyd house paint that comprises anantifungal peptidic agent is as follows in Table 5:

TABLE 5 Component Weight or Volume Grind: first alkyd 232.02 lb or 29.9gallons second alkyd 154.2 lb or 20 gallons aliphatic solvent: duodecane69.55 lb or 1.7 gallons lecithin 7.8 lb or 0.91 gallons TiO₂ 185.25 lbor 5.43 gallons 10 micron silica 59.59 lb or 2.7 gallons bentonite clay18.00 lb or 1.44 gallons second alkyd 97.22 lb or 12.61 gallons firstalkyd 69.84 lb or 9.00 gallons antifungal peptidic agent - effectiveamount/up to optionally, in combination with 7.8 lb or 0.82 gallons aconventional mildewcide Letdown: aliphatic solvent: dudecane) 19.50 lbor 3.00 gallons first drier: 12% solution cobalt) 2.00 lb or 0.23gallons second drier: 18% solution Zr) 2.92 lb or 0.32 gallons thirddrier: 10% solution Ca) 8.00 lb or 0.98 gallons Anti skinning agent:methyl ethyl ketoxime 3.22 lb or 0.42 gallons Aliphatic solvent 9.75 lbor 1.50 gallons

A preferred exterior flat latex house paint comprising an antifungalpeptidic agent will contain the following components, listed in typicalorder of addition in Table 6:

TABLE 6 Component Weight or Volume water 244.5 lb or 29.47 gallonshydroxyethylcellulose 3 lb or 0.34 gallons glycols 60 lb or 6.72 gallonspolyacrylate dispersant 6.8 lb or 0.69 gallons Antifungal Peptidic Agenteffective amount up to optionally, other biocide(s) 10 lb or 1 gallonsnon-ionic surfactant 1 lb or 0.11 gallons titanium dioxide 225 lb or6.75 gallons silicate mineral 160 lb or 7.38 gallons calcined clay 50 lbor 2.28 gallons acrylic latex, @ 60% 302.9 lb or 34.42 gallonscoalescent 9.3 lb or 1.17 gallons defoamers 2 lb or 0.26 gallonsammonium hydroxide 2.2 lb or 0.29 gallons 2.5% HEC solution 76 lb or9.12 gallons antifungal peptidic agent 1.8 lb or 0.82 gallons

From these representative formulations, it will be readily appreciatedthat a wide variety of paints and other coating compositions may beimproved by addition of an antifungal peptidic agent. Some of theseinclude industrial water-borne coating formulations (e.g., air drycoatings, air dry or force air dry coatings, anti-skid of non-slipcoatings, bake dry coatings, clear coatings, coil coatings, concretecoatings, dipping enamels, lacquers, primers, protective coatings, sprayenamels, traffic and airfield coatings); architectural water-bornecoating formulations (e.g., exterior paints, exterior enamels, exteriorcoatings, interior paints, interior enamels, interior coatings,exterior/interior paints, exterior/interior enamels, exterior/interiorprimers, and exterior/interior stains); solvent borne coatingformulations (e.g., exterior paints, exterior enamels, exteriorcoatings, exterior sealers, exterior fillers, exterior primers, interiorpaints, interior enamels, interior coatings, interior primers,exterior/interior paints, exterior/interior enamels, exterior/interiorcoatings, and exterior/interior varnishes); and prepaint specialtiesand/or surface tolerant coatings (e.g., fillers, sealers, rustpreventives, galvanizers, caulks, grouts, glazes, phosphatizers,corrosion inhibitors, neutralizers, graffiti removers and floorsurfacers).

An antifungal paint or coating containing an antifungal peptidic agentmay then be tested and used as described elsewhere herein, or theproduct may be employed for any other suitable purpose as would berecognized by one of skill in the art in light of this disclosure. Forinstance, the physical properties (e.g., purity, density, solubility,volume solids and/or specific gravity, rheology, viscometry, andparticle size) of the resulting antifungal liquid paint or other coatingproduct, can be assessed using standard techniques that are known in theart and/or as described in PAINT AND COATING TESTING MANUAL, 14^(th) ed.of the Gardner-Sward Handbook, J. V. Koleske, Editor (1995), AmericanSociety for Testing and Materials (ASTM), Ann Arbor, Mich., andapplicable published ASTM test methods. Alternatively, any othersuitable testing method as would be known to one of ordinary skill inthe art in light of the present disclosures, may be employed forassessing physical properties of the paint or coating mixture containingan above-described antifungal peptide additive.

EXAMPLE 8 Inhibition of In-Can Mold Growth by Antifungal Peptides

As mentioned in the background discussion, the quality of a liquidcoating mixture may suffer markedly if microorganisms degrade one ormore of the components during storage. Since many of the coatingproducts in use today contain ingredients that make it susceptible orprone to fungal infestation and growth, it is common practice to includea preservative. Although bacterial contamination may be a contributingfactor, fungi are typically a primary cause of deterioration of a liquidpaint or coating. Foul odor, discoloration, thinning and clumping of theproduct, and other signs of deterioration of components render theproduct commercially unattractive and/or unsatisfactory for the intendedpurpose. If the container will be opened and closed a number of timesafter its initial use, in some instances over a period of several monthsor years, it will inevitably be inoculated with ambient fungus organismsor spores subsequent to purchase by the consumer.

To avoid spoilage, it is especially desirable to ensure that the productwill remain stable and usable for the foreseeable duration of storageand use by enhancing the long-term antifungal properties of the paint orcoating with an antifungal peptide agent. The in-can stability andprospective shelf life of a paint or coating mixture containing anabove-described antifungal peptide agent may be assessed using anyappropriate testing method as would be known to one of skill in the artusing conventional microbiological techniques. A fungus known to infectpaints or other coatings is preferably employed as the test organism.

EXAMPLE 9 Testing Protocols for Evaluating Antifungal Coatings

One suitable assay protocol for evaluating coatings containing anantifungal peptide is described by the American Society for Testing andMaterials (ASTM) in D-5590-94 (“Standard Test Method for Determining theResistance of Paint Films and Related Coatings to Fungal Defacement byAccelerated Four-Week Agar Plate Assay”), which is hereby incorporatedherein by reference. The test method is modified as indicated below, andgenerally comprises:

-   -   (a) preparing a set of four 1×10 cm aluminum coupons        approximately 1/32 in thick are prepared as follows: (1) blank        Al coupon; (2) Al coupon coated with an aqueous solution of a        peptide produced and identified as described in the preceding        examples, and allowed to dry; (3) Al coupon coated on both sides        with a base paint composition, allowed to dry, and then the        paint film is coated with a like amount of the same test peptide        solution as applied to coupon 2; and (4) Al coupon painted with        a paint mixture containing the same base paint composition as        for coupon 3 and a like amount of the peptide, as for coupons 2        and 3. Preferably duplicate or triplicate sets of these        specimens are prepared. Optionally, a conventional biocide may        be included as a positive control. The base paint composition        may be any suitable water-based latex paint, without biocides,        which is available from a number of commercial suppliers.    -   (b) Each of the specimens from (a) is placed on a bed of        nutrient agar and uniformly innoculated with a fungal        suspension. A preferred test organism is Fusarium oxysporum. The        fungal suspension may be applied by atomizer or by pipet,        however a thin layer of nutrient agar mixed with the fungal        innoculum is preferred.    -   (c) The specimens are incubated at about 28° C. under 85 to 90%        relative humidity for 4 weeks.    -   (d) Fungal growth on each specimen is preferably rated weekly as        follows: None=0; traces of growth (<10% coverage)=1; light        growth (10-30%)=2; moderate growth (30-60%)=3; and heavy growth        (60% to complete coverage)=4.

Another suitable assay protocol for testing the antifungal properties ofa coating or paint film containing an antifungal peptide is described bythe ASTM in D-5590-94 (“Standard Test Method for Resistance to Growth ofMold on the Surface of Interior Coatings in an Environmental Chamber”),which is hereby incorporated herein by reference. The testing protocolgenerally includes:

-   -   (a) Preparation of the Coated Surface. Duplicate or triplicate        sets of approximately ½ in. thick, 3×4 in. untreated wooden or        gypsum board panels are prepared as follows: (1) blank        panel; (2) coated with an aqueous solution of a peptide produced        and identified as described in the preceding examples, and        allowed to dry; (3) coated on both sides with a base paint        composition, allowed to dry, and then the paint film is coated        with a like amount of the same test peptide solution as applied        to panel 2; and (4) painted with a paint mixture containing the        same base paint composition as for panel 3 and a like amount of        the peptide, as for panels 2 and 3. Optionally, a conventional        biocide may be included as a positive control.    -   (b) Contamination. The panels are randomly arranged and        suspended in an environmental cabinet above moist soil that has        been inoculated with the desired fungus, preferably Fusarium        oxysporum. Enough free space is provided to allow free        circulation of air and avoiding contact between the panels and        the walls of the cabinet.    -   (c) Incubation. The panels are incubated for two weeks at        30.5-33.5° C. and 95-98% humidity. (d) Scoring. A set of panels        (test, control, and, optionally, a positive control) are removed        for analysis at intervals, preferably weekly. The mold growth on        the specimen panels is rated as described above.

Alternatively, one or more equivalent testing protocols may be employed,and field tests of coating compositions containing laboratory-identifiedantifungal peptides or candidate peptides may be carried out inaccordance with conventional methods as would be known to those of skillin the art.

EXAMPLE 10 Latex Paints with Antifungal Peptidic Agents

Both the interior latex (Olympic Premium, flat, ultra white, 72001) andacrylic paints (Sherwin Williams DTM, primer/finish, white, B66W1;136-1500) appeared to be toxic to both Fusarium and Aspergillus.Therefore, eight individual wells (48-well microtito plate) of eachpaint type were extracted on a daily basis with 1 ml of phosphate bufferfor 5 days (1-4 & 6) and then the plates were allowed to dry beforerunning the assay. Each well contained 16 ul of respective paint.

Extract testing: The extract from two wells each of the two paints foreach day was tested for toxicity by mixing the extract 1:1 with 2×medium and inoculating with spores (10E4) of Aspergillus or Fusarium.The extracts had no affect on growth of either test fungus.

Well testing: The extracted and non-extracted wells for each of thepaints were tested with a range of inoculum levels in growth mediumusing the two different fungi. For Fusarium the range was 10E1-10E4 andfor Aspergillus 10E2-10E5.

Well Testing of Acrylic Paint Plates: Both Fusarium and Aspergillus grewin all extracted wells at all inoculum levels. Only Aspergillus grew innon-extracted wells at the 10E5 level and not at lower levels indicativeof an inherent biocidal capability.

Well Testing of Latex Paint Plates: Fusarium grew in the extracted wellsonly at the 10E4 inoculum level but not at 10E1-10E3. Aspergillus grewin all extracted wells showing an inoculum level effect. No growth wasobserved for either Fusarium or Aspergillus in non-extracted wells.

Conclusion: Extraction of the toxic factor(s) found in both paints waspossible. However, it appeared that it may be less extractable from thelatex paint.

Evaluation of peptide activity in presence of acrylic and latex paints:It was established that it was possible to extract both acrylic andlatex paints dried in a 48-well format to make them non-toxic to thetest microorganisms—Fusarium and Aspergillus. Using that information anexperiment was designed to determine the effect the paint has on peptideactivity against two test organisms.

Experimental design: (1.) Coat 48-well plastic plates with 16 μl ofacrylic or latex paint. Dry for two days under hood. (2.) Extractdesignated wells with 1-ml phosphate buffer changing the buffer on adaily basis for 7 days. Control wells were not extracted to confirmpaint toxicity. (3.) Add 20 μl of peptide series in duplicate todesignated dry paint coated wells. Peptide, SEQ ID No. 41, series wereadded in a two-fold dilution series to wells and allowed to dry. Theconcentration of peptide added ranged from 200 μg/20 μl to 1.5 μg/20 μl.

Inoculated paint-coated plates as follows: (1.) Extracted control wellsreceived 180 μl of medium+20 μl of spore suspension (10⁴ spores/20 μl ofmedium). Inoculum was either Fusarium or Aspergillus in each case. (2.)Non-extracted control wells received 180 μl of medium+20 μl of sporesuspension (10⁴ spores/20 μl of medium). (3.) Extract wells with driedpeptide series received 180 μl of medium+20 μl of spore suspension (10⁴spores/20 μl of medium). In duplicate. (4.) Extract wells that did nothave dried peptide series received 160 μl of medium+20 μl of sporesuspension (10⁴/20 μl of medium)+20 μl peptide series as above. Induplicate. (5.) Plates were observed for growth over a 5-day period.

Growth and peptide controls: (1.) Use sterile non-paint coated 48 wellplastic plates. (2.) Growth control wells for each test fungus received180 μl of medium+20 μl of spore suspension (10⁴ spores/20 μl of medium).(3.) Peptide activity controls received 160 μl of medium+20 μl of sporesuspension (10⁴ spores/20 μl of medium)+20 μl peptide series as above.Peptide series were added in a two-fold dilution series to wells andrange from 200 μg/20 μl to 1.5 μg/20 μl. Therefore, the range of peptidetested was 200 μg/200 μl or 1.0 μg/μl (1000 μg/ml) to 0.0075 μg/μl (7.5μg/ml). (4.) Uninoculated medium served as blank for absorbance readingstaken at 24, 48, 72, 96 and 120 h.

Results:

Unextracted wells containing either latex or acrylic paint inhibitedgrowth of both Fusarium and Aspergillus. Extracted wells containingeither latex or acrylic paint allowed growth of both Fusarium andAspergillus.

The calculated MIC for Fusarium in peptide activity control experimentswas 15.62 μg/ml. For Aspergillus the calculated MIC was 61.4 μg/ml.

For extracted acrylic-coated plates the following results were obtained.

Controls as stated in above.

For Fusarium with dried peptide, inhibition was seen at 1000 and 500μg/ml after 5 days. Spores exposed to liquid peptide added to dry paintwells were inhibited at 1000, 500 and 250 μg/ml after 4 days, and 1000and 500 μg/ml after 5 days.

For Aspergillus with dried peptide, inhibition was seen at 1000 μg/mlafter 5 days. Spores exposed to liquid peptide added to dry paint wellswere inhibited at 1000 and 500 μg/ml after 5 days.

For extracted latex-coated plates the following results were obtained.

Controls as stated above.

For Fusarium with dried peptide, inhibition was seen at 1000 μg/ml after5 days. Spores exposed to liquid peptide added to dry paint wells wereinhibited at 1000 μg/ml after 5 days.

For Aspergillus with dried peptide, inhibition was seen at 1000 μg/mlafter 5 days. Spores exposed to liquid peptide added to dry paint wellswere inhibited at 1000 μg/ml after 5 days.

EXAMPLE 11 Coating a Surface to Inhibit Fungus Infestation and Growth

When anchorage, food and moisture are available, fungus microorganismsare able to survive where temperatures permit. Particularly susceptiblesurfaces include porous materials such as stone, brick, wall board(sheetrock) and ceiling tiles; and semi-porous materials, includingconcrete, unglazed tile, stucco, grout, painted surfaces, roofing tiles,shingles, painted or treated wood and textiles. Any type of indoor oroutdoor object, structure or material that is capable of providinganchorage, food and moisture to fungal cells is potentially vulnerableto infestation with mold, mildew or other fungus. Moisture generallyappears due to condensation on surfaces that are at or below the dewpoint for a given relative humidity. To inhibit or prevent fungusinfestation and growth, one or more antifungal peptidic agents fromExample 1 or 3-6, preferably approximately 250-1000 mg/L of thehexapeptide of SEQ ID No. 41, is dissolved or suspended in water andapplied by simply brushing or spraying the solution onto a pre-paintedsurface such as an exterior wall that is susceptible to moldinfestation. Conventional techniques for applying or transferring acoating material to a surface are well known in the art and are suitablefor applying the antifungal peptide composition. The selected peptideshave activity for inhibiting or preventing the growth of one or moretarget fungi. The applied peptide solution is then dried on the paintedsurface, preferably by allowing it to dry under ambient conditions. Ifdesired, drying can be facilitated with a stream of warm, dry air.Optionally, the application procedure may be repeated one or more timesto increase the amount of antifungal peptide that is deposited per unitarea of the surface. As a result of the treatment, when the treatedsurface is subsequently subjected to the target mold organisms or sporesand growth promoting conditions comprising humidity above about typicalindoor ambient humidity, presence of nutrients, and temperature aboveabout typical indoor ambient temperature and not exceeding about 38° C.,the ability of the surface to resistance fungal infestation and growthis enhanced compared to its pre-painted condition before application ofthe antifungal peptide.

A simple spray-coated surface may not provide sufficient durability forcertain applications such as surfaces that are exposed to weathering.Longer-term protection may be provided against adhesion and growth ofmold by mixing one or more of the antifungal peptides with a base paintor other coating composition, which may be any suitable, commerciallyavailable product as are well known in the art. Preferably the basecomposition is free of chemicals and other additives that are toxic tohumans or animals, and/or that fail to comply with applicableenvironmental safety rules or guidelines. The typical components,additives and properties of conventional paints and coating materials,and film-forming techniques, are well known in the art and are alsodescribed in U.S. patent application Ser. No. 10/655,345 filed Sep. 4,2003 and U.S. patent Ser. No. 10/792,516 filed Mar. 3, 2004, which ishereby incorporated herein by reference.

If additional, long-term protection against growth and adhesion of mold,mildew and fungus is desired, the paint or other coating composition mayinclude a barrier material that resists moisture penetration and alsoprevents or deters penetration and adhesion of the microorganisms andthe airborne contaminants which serve as food for the growing organisms.Some typical water repellent components are acrylic, siliconates,metal-stearates, silanes, siloxane and paraffinic waxes. The user willpreferably take additional steps to deter mold infestation includeavoiding moisture from water damage, excessive humidity, water leaks,condensation, water infiltration and flooding, and taking reasonablesteps to avoid buildup of organic matter on the treated surface.

EXAMPLE 12 Method of Treating a Fungus-Infested Surface

Although it is preferred that in situations where existing fungal growthis present, the mold colonies and spores are first removed orsubstantially eliminated before application of one of the presentantifungal coatings, it is expected that in some situations anantifungal compositions will be applied to existing mold infectedsurfaces. In this case, the composition, containing one or moreantifungal peptides, may inhibit, arrest the growth of, or substantiallyeradicate the mold. Early detection and treatment is highly preferred inorder to minimize the associated discoloration or other deterioration ofthe underlying surface due to mold growth. The treatment procedure mayconsist of simply applying one or more coats of an antifungal peptidesolution, paint or other coating composition as described above inExample 11.

EXAMPLE 13 Method of Impregnating a Porous Substrate to Inhibit FungusGrowth

Porous or semi-porous objects or materials such as paper, fabrics,carpet, some types of stone, and many other items that are employedindoors or outdoors, have internal surface areas that can be susceptibleto infestation by mold and are very difficult to treat effectively byconventional methods. It is within the scope of the present invention toimpregnate such porous objects with a coating material containing one ormore antifungal peptide, as described in one or more of the precedingexamples. The liquidity of the composition is such that it is capable ofpenetrating into the pores of the object. In this way, an effectiveamount of the antifungal peptide is deposited on the internal surfacesas well as the exterior ones. Circumstances requiring treatment of aporous surface may benefit from using a relatively thin coating materialrather than a thick, pigmented paint, in order to facilitate penetrationof the pores.

EXAMPLE 14 Coating a Fruit or Grain Storage Vessel to Inhibit Mold

The interior walls of grain silos or other fruit or grain storage ortransportation tanks are coated with a peptidic antifungal compositionof Example 12 or 13 to deter the attachment and growth of mold organismsinside the container. By selecting antifungal peptides that targetspecific organisms, and are non-toxic to humans or animals, moldcontamination of a wide variety of agricultural products may bedeterred.

EXAMPLE 15 Use of Antifungal/Anti-Bacterial Peptides

Over the past decade, outbreaks of food poisoning and hospital-acquiredinfections by so-called “super bugs” have become increasingly frequent.These are strains of bacteria that are resistant to conventionalantibiotics, such as Methicillin-resistant Staphylococcus aureus (MRSA)and Vero-cytotoxin producing variants of Escherichia coli. Worldwidepublic concern about hygienic surfaces have also been heightened todaydue to the emergence and spread of new viral infections such as SARS.The current proliferation of antimicrobial cleaners, utensils, foodpreparation surfaces and coating systems aimed at fulfilling the demandsof an increasingly hygiene conscious public are a testament to thosewidespread concerns.

Some of the antifungal peptides, particularly the 8-10 amino acidresidue long peptides also have the property of inhibiting the growth ofbacteria, including disease-causing bacteria such as Staphalococcus andStreptococcus. Thus, it is known that peptides such as 41, 197, 198, and199, can inhibit growth of E. amylovora, E. carotovora, E. coli, R.solanocerum, S. aureus, and S. faecalis in standard media at IC50's ofbetween 10-1100 mg/ml and MIC's of between 20-1700 mg/ml. Staphalococcusand Streptococcus bacteria are of special concern in hospitalenvironments where antibiotic resistance is increasingly common. Amultipurpose paint or coating is prepared by combining one or moreantifungal peptide selected as described in any of Examples 1-6 with oneor more antibacterial peptide. One such combination is the peptide ofSEQ ID No. 41 and the peptide of SEQ ID No. 41. Alternatively a peptideis selected with both antifungal and antibacterial peptides 6-10. Paintsand other coatings containing the antifungal/antibacterial peptides willbe applied to surfaces to lend antifungal and anti-bacterial propertiesto those surfaces. It is expected that the use of these and otherantifungal/antibacterial peptidic agents will avoid the problem humantoxicity that is associated with conventional biocidal compounds intoday's paints and coatings. The advantage of combined antifungal andantibacterial activity will find particular usefulness in hospitalenvironments and other health care settings.

EXAMPLE 16 Combined Use of Antifungal Peptides and Other Antimicrobials

A paint composition containing one or more conventional antifungalsubstances may be modified by addition of one or more of the antifungalpeptides described herein. As described in preceding examples, theantifungal peptidic agent may be a single peptide of precisely knownsequence, preferably the hexapeptide of SEQ ID No. 198 or anantifungal/antibacterial peptide as described in Example 15.Alternatively, the peptidic agent may be a peptide library aliquotcontaining a mixture of peptides in which at least two (and preferablythree or four) of the N-terminal amino acid residues are known. If thepeptidic agent is a mixture of peptides, one or more peptides will haveantifungal activity.

Combining a non-peptidic antifungal agent with one or more antifungalpeptides may provide antifungal activity over and above that seen witheither the peptide(s) or the non-peptidic agent alone. The expectedadditive inhibitory activity of the combination is calculated by summingthe inhibition levels of each component alone. The combination is thentested on the test organism to derive an observed additive inhibition.If the observed additive inhibition is greater than that of the expectedadditive inhibition, synergy is exhibited. More specifically, asynergistic combination of an antifungal peptide, or an aliquot of apeptide library containing at least one antifungal peptide, occurs whentwo or more fungal cell growth-inhibitory substances distinct from thepeptide or peptide library aliquot are observed to be more inhibitory tothe growth of a test organism than the sum of the inhibitory activitiesof the individual components alone.

A testing method for determining additive or synergistic combinationscomprises first creating a synthetic peptide combinatorial library. Eachaliquot of the library represents an equimolar mixture of peptides inwhich at least the two C-terminal amino acid residues are known. Usingthe testing methods described in one or more of U.S. Pat. No. 6,020,312,U.S. Pat. No. 5,885,782, and U.S. Pat. No. 5,602,097 it is possible todetermine for each such aliquot of the synthetic peptide combinatoriallibrary, a precisely calculated concentration at which it will inhibit atest fungus in a coating. Next, the aliquot of the synthetic peptidecombinatorial library is mixed with at least one non-peptide antifungalcompound to create a test mixture. As with the peptide component of themixture, the baseline ability of the non-peptide antifungal substance toinhibit the test fungus is determined initially. Next, the test fungusis contacted with the test mixture, and the inhibition of growth of thetest organism is measured as compared to at least one untreated control.More controls are desirable, such as a control for each individualcomponent of the mixture. Similarly, where there are more than twocomponents being tested, the number of controls to be used must beincreased in a manner well known to those of skill in the art of growthinhibition testing. From the separate test results for the peptidic andnon-peptidic agents the expected additive effect on inhibition of growthis determined using standard techniques. After the growth inhibitiontests are complete for the combination of peptidic and non-peptidicagents, the actual or observed effect on the inhibition of growth isdetermined. The expected additive effect and the observed effect arethen compared to determine whether a synergistic inhibition of growth ofthe test fungus has occurred. The methods used to detect synergy mayutilize non-peptide antimicrobial agents in combination with theinhibitory peptides described above.

As described above, an antifungal peptidic agent may be used incombination with one or more existing fungicides and/or fungistaticsidentified herein or as would be known to one of ordinary skill in theart. It is expected that some combinations of an antifungal peptidicagent with another fungicide and/or fungistatic may provide advantagessuch as a broader range of activity against various organisms, asynergistic antifungal or preservative effect, or a longer duration ofeffect.

Another potentially advantageous combination includes an antifungalpeptidic agent and a preservative that acts against non-fungal organisms(e.g., a bactericide, an algaecide), as it is contemplated that manyfungal prone compositions and surfaces coated with such compositions arealso susceptible to damage by a variety of organisms. Examples ofpreservatives that an antifungal peptidic agent may substitute forand/or be combined include, but are not limited to those non-peptidicantimicrobial compounds (i.e., biocides, fungicides, algaecides andmildewcides) have been shown to be of utility and are currentlyavailable and approved for use in the U.S./NAFTA, Europe, and the AsiaPacific region. These antimicrobial agents are listed in Table 4,together with the name of a supplier.

Certain peptides contemplated for use as described herein have beenshown (in one or more of U.S. Pat. Nos. 6,020,312; 5,602,097; and5,885,782) to involve synergy between antifungal peptides andnon-peptide antifungal agents that is useful in controlling growth ofthe Fusarium, Rhizoctonia, Ceratocystis, Pythium, Mycosphaerella,Aspergillus and Candida genera of fungi. In particular, synergisticcombinations have been described and successfully used to inhibit thegrowth of Aspergillus fumigatus and A. paraciticus, and also Fusariumoxysporum with respect to agricultural applications. It is expected thatthese and other synergistic combinations of peptide and non-peptideagents will be useful as additives in paints, coatings and othercompositions for deterring, preventing, or treating a fungalinfestations.

EXAMPLE 17 Combined Use of Antifungal Peptides and OP Degrading Agents

Beyond the concerns about food poisoning and hospital acquiredinfections by antibiotic-resistant “super bugs,” and worries aboutSARS-like outbreaks, there is also a need to prevent or protect againstthe possibility of contamination of public facilities and surfaces bytoxic chemicals due to accidental spills, improper application ofcertain insecticides, or as a result of deliberate criminal orterroristic acts. In particular, organophosphorus compounds(“organophosphate compounds” or “OP compounds”) and organosulfur (“OS”)compounds, which are used extensively as insecticides, are highly toxicto many organisms, including humans. OP compounds function as nerveagents, and some of the most toxic OP compounds are known to have beenused as chemical warfare agents. As discussed in more detail incopending U.S. patent application Ser. No. 10/655,345 filed Sep. 4,2003, some OP chemical warfare agents can be taken up through skincontact and can remain on material, equipment and terrain for longperiods of time (e.g., weeks). By addition of a thickener (e.g., avariety of carbon polymers), even volatile OP agents may be renderedless volatile and more persistent on a contaminated surface.

Thus, it can be readily appreciated that in some situations amultifunctional surface treatment that combines antifungal propertieswith the ability to degrade organophosphorus compounds would bedesirable. Such composition may be in the form of a coating, a paint, anon-film forming coating, an elastomer, an adhesive, an sealant, amaterial applied to a textile, or a wax, and may be modified by additionof one or more antifungal peptide selected as described in Examples 1-6and an organophosphorus compound detoxifying agent such as an OPdegrading enzyme or cellular material containing such activity. SuitableOP degrading agents are described in copending U.S. patent applicationSer. No. 10/655,345 filed Sep. 4, 2003 and U.S. patent application Ser.No. 10/792,516 filed Mar. 3, 2004 and hereby incorporated herein byreference.

EXAMPLE 18 Adhesives, Sealants and Elastomers Containing AntifungalPeptides

The antifungal additives described above are expected to be additionallyuseful for coating or mixing into sealants and elastomers such as groutsand caulks, especially those that are in frequent contact with, orconstantly exposed to fungal nutrients and/or moisture. Examples ofadhesives and sealants (e.g., caulks, acrylics, elastomers, phenolicresin, epoxy, polyurethane, anaerobic and structural acrylic,high-temperature polymers, water-based industrial type adhesives,water-based paper and packaging adhesives, water-based coatings, hotmelt adhesives, hot melt coatings for paper and plastic, epoxyadhesives, plastisol compounds, construction adhesives, flockingadhesives, industrial adhesives, general purpose adhesives, pressuresensitive adhesives, sealants, mastics, urethanes) for various surfaces(e.g., metal, plastic, textile, paper), and techniques of preparationand assays for properties, have been described in Skeist, I., ed.,Handbook of Adhesives, 3rd Ed., Van Nostrand Reinhold, New York, 1990;Satriana, M. J. Hot Melt Adhesives: Manufacture and Applications, NoyesData Corporation, New Jersey, 1974; Petrie, E. M., Handbook of Adhesivesand Sealants, McGraw-Hill, New York, 2000; Hartshorn, S. R., ed.,Structural Adhesives—Chemistry and Technology. Plenum Press, New York,1986; Flick, E. W., Adhesive and Sealant Compound Formulations, 2nd Ed.,Noyes Publications, New Jersey, 1984; Flick, E., Handbook of RawAdhesives 2nd Ed., Noyes Publications, New Jersey, 1989; Flick, E.,Handbook of Raw Adhesives, Noyes Publications, New Jersey, 1982;Dunning, H. R., Pressure Sensitive Adhesives—Formulations andTechnology, 2nd Ed., Noyes Data Corporation, New Jersey, 1977; andFlick, E. W., Construction and Structural Adhesives and Sealants, NoyesPublications, New Jersey, 1988. An adhesive, sealant or elastomercomposition containing one or more conventional antifungal substance maybe modified by addition of one or more of the antifungal peptidesdescribed in Examples 1-6. The antifungal peptidic agent may be a singlepeptide of precisely known sequence, preferably the hexapeptide of SEQID No. 41 or an antifungal/antibacterial peptide as described in Example14. Alternatively, the peptidic agent may be a peptide library aliquotcontaining a mixture of peptides in which at least two (and preferablythree or four) of the N-terminal amino acid residues are known. If thepeptidic agent is a mixture of peptides, at least one peptide will haveantifungal activity.

EXAMPLE 19 Antifungal Textile Finish

An antifungal peptidic agent may also be incorporated into a materialapplied to a textile, such as, for example, a textile finish. Textilefinishes (e.g., soil-resistant finishes, stain-resistant finishes) andrelated materials for application to a textile are described, forexample, in Johnson, K., ANTISTATIC COMPOSITIONS FOR TEXTILES ANDPLASTICS, Noyes Data Corporation, New Jersey, 1976; Rouette, H. K.,ENCYCLOPEDIA OF TEXTILE FINISHING, Springer, Verlag, 2001; TEXTILEFINISHING CHEMICALS: AN INDUSTRIAL GUIDE, by Ernest W. Flick, NoyesPublications, 1990; and HANDBOOK OF FIBER FINISH TECHNOLOGY, by PhilipE. Slade, Marcel Dekker, 1998. One type of water repellent and/or oilrepellent textile finish is Scotchguard™ (3M Corporate Headquarters,Maplewood, Minn., U.S.A.). A textile finish may be modified by additionof one or more of the antifungal peptides described in Examples 1-6. Theantifungal peptidic agent may be a single peptide of precisely knownsequence, preferably the hexapeptide of SEQ ID No. 41 or anantifungal/antibacterial peptide as described in Example 15.Alternatively, the peptidic agent may be a peptide library aliquotcontaining a mixture of peptides in which at least two (and preferablythree or four) of the N-terminal amino acid residues are known. If thepeptidic agent is a mixture of peptides, at least one peptide will haveantifungal activity.

EXAMPLE 20 Polymer-Linked Antifungal Peptides

In Example 4, above, conjugation of a peptide to a polymer carriermolecule or insoluble substrate is described for stabilizing theantifungal activity in the paint film or coating. That capability mayalso be used to advantage by chemically linking or otherwise associatingone or more antifungal peptides to a polymeric material or plasticfabric which would otherwise be more susceptible to infestation,defacement or deterioration by fungus. Conventional techniques forlinking the N- or C-terminus of a peptide to a long-chain polymer may beemployed. The antifungal peptide may include additional amino acids onthe linking end to facilitate linkage to the PVC polymer. A PVC-membranesuch as a flexible or retractable roof or covering for an outdoorstadium is treated to chemically link antifungal peptides to at least aportion of the outer surface of the membrane prior to its installation.Where an installed polymer membrane covering is already infested bymold, and it is not practical for it to be removed and replaced by anantifungal peptide-linked polymer membrane, it may be feasible to cleanthe existing infestation or discoloration, and then apply or bond asuitable antifungal coating containing a stabilized antifungal peptide.PVC is only one of many well-known types of plastic orpolymer-containing materials that could be linked to an antifungalpeptide in this manner.

EXAMPLE 21 Kit for Preparing an Antifungal Coating

For ease of production, in most instances an antifungal paint or coatingproduct containing antifungal peptidic agents will be provided to theconsumer as a single premixed formulation. Alternatively, in order tooptimize the initial activity and extend the useful lifetime of theantifungal coating, the antifungal peptidic agent may instead bepackaged separately from the paint or coating product into which theantifungal agent is to be added. For increased stability, the peptidicagent may also contain a suitable solid or liquid carrier. As inpreceding examples, the antifungal peptidic agent may comprise one ormore “pure” antifungal peptides of defined sequence, or it may include apeptide library aliquot containing a mixture of peptides in which atleast two (and preferably three or four) of the N-terminal amino acidresidues are known (as in SEQ ID Nos. 1-24). If the peptidic agent is amixture of peptides, at least one will have antifungal activity.

In some situations it may also be preferred to store a fungal-pronematerial in a separate container (“pot”) prior to application, in orderto minimize the occurrence of fungal contamination prior to use and forother reasons. Separation of conventional coating components istypically done to reduce film formation during storage for certain typesof coatings. Accordingly, some or all of the different components of theantifungal composition are stored in a plurality of containers, or as amulti-pack kit, and the components are admixed prior to and/or duringapplication. For example, 0.001% to 100%, including all intermediateranges and combinations thereof, of the antifungal peptidic agent may bestored in a separate container from one or more fungal-prone materialsof the final composition. A multi-pack kit may include one or more potsof a fungal-prone material, preferably including 2- to 5-packs offungal-prone material. A new antifungal composition may be prepared ator near the time of use by combining a fungal-prone material (e.g.,carbon polymer-containing binder) with other coating components,including an antifungal peptide, polypeptide or protein, as describedherein.

DEFINITIONS

The terms used herein have their customary and usual meanings, and areintended to encompass at least the following definitions, consistentwith their use elsewhere herein:

As used herein other than the claims, the terms “a”, “an”, “the” and“said” means “at least one” or “one or more.”

As used herein in the claim(s), when used in conjunction with the words“comprises” or “comprising,” the words “a”, “an”, “the” or “said” mayrefer to one or more than one. As used herein “another” may mean atleast a second or more.

“Fungus” includes multicellular and unicellular organisms in the fungusfamily, including the true fungi, molds, mildews and yeasts. “Mold” issometimes used herein as a synonym for fungi, where the context permits,especially when referring to indoor contaminants. However, the term“mold” also, and more specifically, denotes certain types of fungi. Forexample, the plasmodial slime molds, the cellular slime molds, watermolds, and the everyday common mold. True molds are filamentous fungiconsisting of the mycelium, specialized, spore-bearing structures calledconidiophores, and conidia (spores). “Mildew” is another common name forcertain fungi, including the powdery mildews and the downy mildews.“Yeasts” are unicellular members of the fungus family. For the purposesof the present disclosure, where any of the terms fungus, mold, mildewand yeast is used, the others are implied where the context permits.

“Building materials” include, but are not limited to, conventional andnon-conventional indoor and outdoor construction and decorativematerials, such as wood, sheet-rock (wallboard), paper or vinyl coatedwallboard, fabrics (textiles), carpet, leather, ceiling tiles, celluloseresin wall board (fiberboard), stone, brick, concrete, unglazed tile,stucco, grout, painted surfaces, roofing tiles, shingles, and othermaterials that are cellulose-rich, or are capable of providing nutrientsto fungi, or are capable of harboring nutrient materials and supportingfungal infestation.

“Bioactive” means having an effect on a living organism, especiallyfungal cells, when the context allows.

An “antifungal peptide” refers specifically to a contiguous amino acidsequence from 3 to 100 amino acid residues in length, including allintermediate ranges, and which is capable of exerting antifungalactivity, as defined above. For simplicity, where the context permits,the term “antifungal peptide” also refers to antifungal polypeptides(i.e., a contiguous amino acid sequence from 101 to 10,000 amino acidresidues in length, including all intermediate ranges, and antifungalproteins which are proteinaceous molecules having a contiguous aminoacid sequence of more than 10,000 amino acid residues length. Preferablysuch peptides, polypeptides and proteins are not encoded by the genomeof an organism.

“Antifungal peptidic agent” refers to a peptide, polypeptide or proteinhaving the ability to inhibit the growth of one or more genera and/orspecies of fungi. It is also intended to encompass mixtures of suchpeptides, polypeptides and proteins, together with any associatedstabilizers, carriers, and inactive peptides/polypeptides/proteins.Where the context allows, the term “antifungal peptidic agent” may alsorefer to a peptide library aliquot containing a mixture of peptides inwhich at least two of the N-terminal amino acid residues are known. Ifthe peptidic agent is a mixture of peptides, at least one will haveantifungal activity.

“Antifungal activity” refers to inhibition of fungal cell attachmentand/or growth, and is may also refer to fungal cell killing, as thecontext permits. Accordingly, some antifungal peptidic agents can alsobe denoted as “fungistatic agents” or “fungicides.”

“Inhibition of fungal growth” refers to cessation or reduction of fungalcell proliferation, and can also include inhibition of expression ofcellularly produced proteins in static fungal cell colonies. Suchinhibition can provide or facilitate disinfection, decontamination orsanitization of inanimate objects, which refer to the process ofreducing the number of fungus microorganisms to levels that no longerpose a threat (e.g., to property or human health). Use of a bioactiveantifungal agent can be accompanied by manual removal ofmold-contaminated building materials, in some instances.

The term “biocide” as used herein refers to a substance that killsmicroorganisms and their spores. Depending on the type of microorganismkilled, a biocidal substance may be further defined as a bactericide,fungicide, or algaecide. The term “biostatic” refers to a substance thatprevents the growth of the microorganism and its spores, and encompassesbacteristatic, fungistatic and algaestatic compounds.

A “fungicide” is a biocidal substance used to kill or inactivate aspecific microbial group, the fungi. The term “fungistatic,” is used todenote substances that prevent fungal microorganisms from growing orreproducing, but do not result in substantial inactivation or killing.

An “effective amount” refers to a concentration of antifungal peptidethat is capable of exerting the desired antifungal effect, as definedabove.

An “inanimate object” refers to structures and objects other than livingorganisms. Examples of inanimate objects are architectural structureshaving painted or unpainted surfaces such as the exterior and interiorwalls of buildings, industrial equipment, outdoor sculptures andfurniture, construction materials for indoor or outdoor use, such aswood, stone, brick, wall board (sheetrock), ceiling tiles, concrete,unglazed tile, stucco, grout, roofing tiles, shingles, painted ortreated wood, synthetic composite materials, leather and textiles.

A “base” or “substrate” refers to any surface that can potentiallysupport the infestation and/or growth of a fungus or spore underfavorable conditions for such infestation or growth. It is intended toinclude exterior surfaces of objects as well as interior surfaces ofporous and semiporous objects (e.g., high surface area porous stonestructures), constitutes a surface on which a coating can be directlyapplied and/or impregnated.

The term “coating” has its usual meaning and specifically includes theprocess of applying (e.g., brushing, dipping, spreading, spraying) orotherwise producing a coated surface, which may also be referred to as acoating, coat, covering, film or layer on a surface.

Where the context so indicates, the term “coating” may instead refer tothe coating composition or mixture that is applied. For example, acoating composition may be capable of undergoing a change from a fluentto a nonfluent condition by removal of solvents, vehicles or carriers,by setting, by chemical reaction or conversion, or by solidificationfrom a molten state. The coating or film that is formed may be hard orsoft, elastic or inelastic, permanent or transitory. Where the contextallows, the act of coating also includes impregnating a surface orobject by causing a coating material to extend or penetrate into theobject, or into the interstices of a porous, cellular or foraminousmaterial. The general composition and properties of conventional coatingmaterials are described in U.S. patent application Ser. No. 10/655,345filed Sep. 4, 2003, which is hereby incorporated herein by reference.Additionally, the use of the term “coating” (“coat,” “surface coat,”“surface coating”) is also intended to be consistent with its use inPAINT and Coating Testing Manual, Fourteenth Edition of theGardner-Sward Handbook (Koleske, J. V. Ed.), p. 696, 1995; and in “ASTMBook of Standards, Volume 06.01, Paint—Tests for Chemical, Physical, andOptical Properties; Appearance,” D16-00, 2002, i.e., “a liquid,liquefiable or mastic composition that is converted to a solidprotective, decorative, or functional adherent film after application asa thin layer.” Examples of a coating include a clear coating and apaint.

A “paint” generally refers to a “pigmented liquid, liquefiable or masticcomposition designed for application to a substrate in a thin layerwhich is converted to an opaque solid film after application. Used forprotection, decoration or identification, or to serve some functionalpurpose such as the filling or concealing of surface irregularities, themodification of light and heat radiation characteristics, etc,” [Paintand Coating Testing Manual, Fourteenth Edition of the Gardner-SwardHandbook (Koleske, J. V. Ed.), p. 696, 1995]. Surface treatments,particularly coatings and paints, have been described in U.S. patentapplication Ser. No. 10/655,345 filed Sep. 4, 2003.

“Elastomers” or rubbers are polymers that can undergo large, butreversible, deformations upon a relatively low physical stress.Elastomers (e.g., tire rubbers, polyurethane elastomers, polymers endingin an anionic diene, segmented polyerethane-urea copolymers, dienetriblock polymers with styrene-alpha-methylstyrene copolymer end blocks,poly(p-methylstyrene-b-p-methylstyrene), polydimethylsiloxane-vinylmonomer block polymers, chemically modified natural rubber, polymersfrom hydrogenated polydienes, polyacrylic elastomers, polybutadienes,trans-polyisoprene, polyisobutene, cis-1,4-polybutadiene, polyolefinthermoplastic elastomers, block polymers, polyester thermoplasticelastomer, thermoplastic polyurethane elastomers) and techniques ofelastomer synthesis and elastomer property analysis have been described,for example, in Walker, B. M., ed., Handbook of ThermoplasticElastomers, Van Nostrand Reinhold Co., New York, 1979; Holden, G., ed.,et. al., Thermoplastic Elastomers, 2nd Ed., Hanser Publishers, Verlag,1996.

An “adhesive” is a composition that is capable of uniting, bonding orholding at least two surfaces together, preferably in a strong andpermanent manner (e.g., glue, cement, paste).

A “sealant” is a composition capable of attaching to at least twosurfaces, filling the space between them to provide a barrier orprotective coating (e.g., by filling gaps or making a surfacenonporous).

A “fungal-prone material” is a substance that is capable of serving as afood source for a fungus, or is a material that contains one or moresuch substance. For example, in the context of a paint or coatingcomposition, a fungal-prone material may be a binder containing acarbon-based polymer that serves as a nutrient for a fungus.

All patents, published patent applications and other publications citedherein are hereby incorporated herein by reference to the extent thatthey describe materials and methods supplementary to that set forthherein. One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out any objects and obtain the endsand advantages mentioned as well as those inherent therein. Thepreferred antifungal compositions and methods described herein areexemplary and intended to be representative of other embodiments whichwill be apparent to those skilled in the art in light of the presentdisclosure. For instance, in light of the present disclosure andrepresentative examples, changes in the disclosed compositions andmethods and other uses will occur to those skilled in the respectivearts of preparing and using paints and coatings, textile finishes,waxes, elastomers, adhesives and sealants which are encompassed withinthe spirit of the invention and defined by the scope of the appendedclaims. The present examples, therefore, are not to be considered aslimiting the scope of the present invention.

1. A bioactive surface coating composition comprising a peptidic agentin a sufficient amount to inhibit the growth of a fungus, bacterium, orboth on an inanimate surface coated with the bioactive surface coatingcomposition, wherein the peptidic agent consists essentially of acontiguous amino acid sequence selected from the group consisting of SEQID Nos. 159-168 and functionally equivalent amino acid substitutedsequences having no more than a +/−2 difference in hydropathic indexvalue of the Kyte-Doolittle scale relative to SEQ ID Nos. 159-168, andwherein the bioactive surface coating composition is a liquid,liquefiable or mastic composition that is converted to a solidprotective, decorative, or functional adherent film after application asa thin layer.
 2. The bioactive surface coating composition of claim 1,wherein the peptidic agent is non-cyclic.
 3. The bioactive surfacecoating composition of claim 1, wherein each amino acid of the peptidicagent is independently selected from the group consisting of theD-stereoisomer and L-stereoisomer forms of the amino acid.
 4. Thebioactive surface coating composition of claim 1, wherein the peptidicagent comprises a modified peptide functional group.
 5. The bioactivesurface coating composition of claim 4, wherein the modified peptidefunctional group comprises acetylation of a hydroxyl group, acylation ofa charged lysine residue, acylation of an amino group, acylation of thecarboxyl group of a glutamic acid, acylation of the carboxyl group of anaspartic acid, alkylation of an amino group, amidation of a carboxylgroup, amidation of an amino group, arylation of an amino group,deamidation of an amide group of an asparagine, deamidation of an amidegroup of an glutamine, esterification of a carboxyl group, halogenationof a phenolic group of a tyrosine, nitration of a phenolic group of atyrosine, reduction of a carboxyl group to an alcohol, blockage of aside chain, or attachment of a side chain to a synthetic resin.
 6. Thebioactive surface coating composition of claim 1, wherein the peptidicagent is encapsulated.
 7. The bioactive surface coating composition ofclaim 1, wherein the peptidic agent is linked, tethered, or conjugatedto a carrier molecule.
 8. The bioactive surface coating composition ofclaim 7, wherein the carrier molecule comprises polyethyleneglycol,polyvinylpyrrolidone, sand, a silicate, polystyrene, cellulose, orpolyvinylchloride.
 9. The bioactive surface coating composition of claim1, wherein a weight concentration of the peptidic agent within thebioactive surface coating composition comprises 0.0001% to 20.0%. 10.The bioactive surface coating composition of claim 1, wherein thebioactive surface coating composition is an architectural coating, anindustrial coating, a specification coating, or a combination thereof.11. The bioactive surface coating composition of claim 1, wherein thebioactive surface coating composition is a water-borne coatingcomposition.
 12. A bioactive surface coating composition comprising apeptidic agent in a sufficient amount to inhibit the growth of a fungus,bacterium, or both on an inanimate surface coated with the bioactivesurface coating composition, wherein the peptidic agent consistsessentially of a contiguous amino acid sequence selected from the groupconsisting of SEQ ID Nos. 159-168 in the order of their C-terminus totheir N-terminus and functionally equivalent amino acid substitutedsequences having no more than a +/−2 difference in hydropathic indexvalue of the Kyte-Doolittle scale relative to SEQ ID Nos. 159-168 in theorder of their C-terminus to their N-terminus, and wherein the bioactivesurface coating composition is a liquid, liquefiable or masticcomposition that is converted to a solid protective, decorative, orfunctional adherent film after application as a thin layer.
 13. Thebioactive surface coating composition of claim 12, wherein the peptidicagent is non-cyclic.
 14. The bioactive surface coating composition ofclaim 12, wherein each amino acid of the peptidic agent is independentlyselected from the group consisting of the D-stereoisomer andL-stereoisomer forms of the amino acid.
 15. The bioactive surfacecoating composition of claim 12, wherein the peptidic agent comprises amodified peptide functional group.
 16. The bioactive surface coatingcomposition of claim 12, wherein the peptidic agent is encapsulated. 17.The bioactive surface coating composition of claim 12, wherein thepeptidic agent is linked, tethered, or conjugated to a carrier molecule.18. The bioactive surface coating composition of claim 17, wherein thecarrier molecule comprises polyethyleneglycol, polyvinylpyrrolidone,sand, a silicate, polystyrene, cellulose, or polyvinylchloride.
 19. Thebioactive surface coating composition of claim 12, wherein a weightconcentration of the peptidic agent within the bioactive surface coatingcomposition comprises 0.0001% to 20.0%.
 20. The bioactive surfacecoating composition of claim 12, wherein the bioactive surface coatingcomposition is a water-borne coating composition.